49 human secreted proteins

ABSTRACT

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.

This application is a continuation application of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, now abandoned which is a continuation of Ser. No. 09/511,554 filed Feb. 23, 2000, now abandoned which is a continuation-in-part of, and claims benefit under 35 U.S.C. §120 of copending International patent application Serial No. PCT/US99/19330, filed Aug. 24, 1999, and published in the English language, which is hereby incorporated by reference, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application Nos. 60/097,917 filed Aug. 25, 1998 and 60/098,634 filed Aug. 31, 1998.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides, and their production.

BACKGROUND OF THE INVENTION

Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eucaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals,” which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.

Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a “linker” holding the protein to the membrane.

Despite the great progress made in recent years, only a small number of genes encoding human secreted proteins have been identified. These secreted proteins include the commercially valuable human insulin, interferon, Factor VIII, human growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in light of the pervasive role of secreted proteins in human physiology, a need exists for identifying and characterizing novel human secreted proteins and the genes that encode them. This knowledge will allow one to detect, to treat, and to prevent medical diseases, disorders, and/or conditions by using secreted proteins or the genes that encode them.

SUMMARY OF THE INVENTION

The present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

DETAILED DESCRIPTION

Definitions

The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a generic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection (“ATCC”). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/mil salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences identified by an integer specified in Table 1.

“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)

Polynucleotides and Polypeptides of the Invention

Features of Protein Encoded by Gene No: 1

The translation product of this gene shares sequence homology with the TAP2 of Rattus norvegicus (See, e.g., Genbank Accession No.gi|407479) AAD15830; all references available through this accession are hereby incorporated by reference herein.) which is thought to be important in antigen presentation in T-cells. As such, the protein product of this gene may be useful for a variety of diagnostic tests for various immune system dysfunctions, and for intervention of the antigen presentation process (e.g., to enhance the immune response to vaccines and diminish the immune response associated with autoimmune disease). The major histocompatibility complex (Mhc) regions of mice, rats, and humans all contain a pair of related genes, TAP1 and TAP2, which encode members of a large superfamily of proteins of similar structure and function. A functional TAP1/TAP2 heterodimer is probably required for efficient presentation of antigens to CD8(+) T cells. This heterodimer resides in the membrane of the endoplasmic reticulum, and transports peptides from the cytoplasm into the endoplasmic reticulum lumen for binding to Mhc class I molecules.

The translation product of this gene also shares sequence homology with the transport-associated proteins and ATP-binding proteins (see, e.g., Genbank accesssion numbers CAB05918 (z83328.1) and AAB95060 (AF040659.1); all references available through these accessions are hereby incorporated by reference herein.)

Preferred polypeptides of the invention comprise the following amino acid sequence:

EPHRGPHLPPDLGHHHGQRPGLQNINVFLRNTVKVTGVVVFMFSLSWQLSLV (SEQ ID NO: 123) TFMGFPIIMMVSNIYGKYYKRLSKEVQNALARASNTAEETISAMKTVRSFAN EEEEAEVYLRKLQQVYKLNRKEAAAYMYYVWGSGLTLLVVQVSILYYGGH LVISGQMTSGNLIAFIIYEFVLGDCMENVSFSLSPGKVTALVGPSGSGKSSCVN ILENFYPLEGGRVLLDGKPISAYDHKYLHRVISLVSQEPVLFARSITDNISYGLP TVPFEMVVEAAQKANAHGFIMELQDGYSTETGEKGAQLSGGQKQRVAWPG LWCGTPQSSSWMKPPALWMPRASI, MSSATWTAASWRTSATSTSLTRCWISGQPACTAAACCWGATIGVAKNSALG (SEQ ID NO: 124) PRRLRASWLVITLVCLFVGIYAMVKLLLFSEVRRPIRDPWFWALFVWTYISLG ASFLLWWLLSTVRPGTQALEPGAATEAEGFPGSGRPPPQASGATLQKLLSYT KPDVAFLVAASFFLIVAALGETFLPYYTGRAIDGIVIQKSMDQFSTAVVIVCLL AIGSSFAAGIRGGIFTLIFARLNIRLRNCLFRSLVSQETSFFDENRTGDLISRLTS DTTMVSDLVSRTSMSSCGTQSRSRAWWSSCSASHGSSPWSPSWASPSS, or HLLRPAHCAFRDGGGGRTEGQCPRLHHGTPGRLQHRDRGEGRPAVRWPEAA (SEQ ID NO: 125) GGMARALVRNPPVLILDEATSALDAESEYLIQQAIHGNLQKHTVLIIAHRLST VEHAHLIVVLDKGRVVQQGTHQQLLAQGGLYAKLVQRQMLGLQPAADFTA GHNEPVANGSHKA.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 12. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 12.

This gene is expressed primarily in brain and testes, and to a lesser extent in amniotic cells, merkel cells and fetal tissue.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neural, reproductive, or immune disorders, particularly immunodeficiency, infection, lymphomas, auto-immunities, cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, neural, reproductive, developmental, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 67 as residues: His-65 to Cys-73, His-144 to Gly-152. Polynucleotides encoding said polypeptides are also provided.

The major histocompatibility complex (Mhc) regions of mice, rats, and humans all contain a pair of related genes, TAP1 and TAP2, which encode members of a large superfamily of proteins of similar structure and function. A functional TAP1/TAP2 heterodimer is probably required for efficient presentation of antigens to CD8(+) T cells. Furmermore, the tissue distribution in merkel cells and homology to TAP indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of conditions in which antigen presentation is at issue, such as general microbial infection, auto-immunity, inflammation or cancer. Alternatively, expression within brain tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection/treatment of neurodegenerative disease states, behavioural disorders, or inflamatory conditions such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered bahaviors, including disorders in feeding, sleep patterns, balance, and preception. In addition, the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3275 of SEQ ID NO:11, b is an integer of 15 to 3289, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:11, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 2

The translation product of this gene shares sequence homology with a human retrovirus related env glycoprotein (for examples, see Genbank Accession No. AAD34324 and AAD14546; all references available through this accession are hereby incorporated herein by reference; for example Lindeskog, M., et al. Virology 258 (2), 441-450 (1999) and Blond, J. L., et al., J. Virol. 73 (2), 1175-1185 (1999)). This similarity indicates that the human protein described herein is cell surface protein.

Preferred polypeptides of the invention comprise the following amino acid sequence:

RLTKTISFSLQNQTAFINSLAKTPYQALTGAALAGSYPIWENENTLSWYLPSPT (SEQ ID NO: 126) TLLSPPVLFCVIQLIFXLPANWSGTCTLVFQAPTINILPPNQTILISVEASISSSPIR NKWALHLITLLTGLGITAALGTGIAGITTSITSYQTLFTTLSNTVEDMHTSITSL QRQLDFLVGVILQNWRVLDLLTTEKGGTCIYLQEECCFCVNESGIVHIAVRRL HDRAAEL, YPIWENENTLSWYLPSPTTLLSPPVLFCV, (SEQ ID NO: 127) or, RVLDLLTTEKGGTCIYLQEECCFCVNE. (SEQ ID NO: 128)

Polynucleotides encoding these polypeptides are also provided.

Further, the gene encoding the disclosed cDNA is believed to reside on the X chromosome. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for the X chromosome.

This gene is expressed primarily in human prostate cancer (stage C fraction), subtracted kidney cortex, adult brain and breast, and to a lesser extent in a variety of normal and transformed tissue types.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, anti-viral therapies, including cancer and other proliferative diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the prostate, kidney, brain and breast, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, neural, endocrine, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 68 as residues: Ser-28 to Ser-37, Ser-50 to Ser-58. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in various transformed tissues, combined with its similarity to retroviral env proteins indicates that the protein product is useful for the detection, diagnosis, and treatment of a variety of cancers and other proliferative disorders. This gene may show utility in gene therapy applications in viral prophylaxis. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2328 of SEQ ID NO:12, b is an integer of 15 to 2342, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:12, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 3

The translation product of this gene shares sequence homology with Cytochrome P450 monooxygenase which is thought to be important in NADPH-dependent oxidation of a number of cellular substrates (See Genbank Accession Nos. gi|1185452, gb|AAC50370.1, and gb|AAB87635.1, in addition to Geneseq Accession No. R72378; all information and references available through these accessions are hereby incorporated herein by reference). The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 11-27 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 28 to 501 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins. Included in this invention as a preferred domain is the cytochrome P450 cysteine heme-iron ligand signature domain, which was identified using the ProSite analysis tool (Swiss Institute of Bioinformatics). Cytochrome P450's [1, 2, 3] are a group of enzymes involved in the oxidative metabolism of a high number of natural compounds (such as steroids, fatty acids, prostaglandins, leukotrienes, etc) as well as drugs, carcinogens and mutagens. Based on sequence similarities, P450's have been classified into about forty different families [4,5]. P450's are proteins of 400 to 530 amino acids; the only exception is Bacillus BM-3 (CYP102) which is a protein of 1048 residues that contains a N-terminal P450 domain followed by a reductase domain. P450's are heme proteins. A conserved cysteine residue in the C-terminal part of P450's is involved in binding the heme iron in the fifth coordination site. From a region around this residue, we developed a ten residue signature specific to P450's. The concensus pattern is as follows: [FW]-[SGNH]-x-[GD]-x-[RHPT]-x-C-[LIVMFAP]-[GAD] [C is the heme iron ligand].

Preferred polypeptides of the invention comprise the following amino acid sequence: FSLGRRHCLG (SEQ ID NO: 129). Polynucleotides encoding these polypeptides are also provided. Further preferred are polypeptides comprising the cytochrome P450 cysteine heme-iron ligand signature domain of the sequence referenced in Table for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence. The additional contiguous amino acid residues may be N-terminal or C-terminal to the cytochrome P450 cysteine heme-iron ligand signature domain. Alternatively, the additional contiguous amino acid residues may be both N-terminal and C-terminal to the cytochrome P450 cysteine heme-iron ligand signature domain, wherein the total N- and C-terminal continuous amino acid residues equal the specified number. The above preferred polypeptide domain is characteristic of a signature specific to cytochrome P450 cysteine heme-iron proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with cytochrome P450 cysteine heme-iron proteins. Such activities are known in the art, some of which are described elsewhere herein. The following references were reference above and are hereby incorporated by reference herein: [1] Nebert D. W., Gonzalez F. J. Annu. Rev. Biochem. 56:945-993(1987). [2] Coon M. J., Ding X., Pemecky S. J., Vaz A. D. N. FASEB J. 6:669-673(1992). [3] Guengerich F. P. J. Biol. Chem. 266:10019-10022(1991).[4] Nelson D. R., Kamataki T., Waxman D. J., Guengerich F. P., Estrabrook R. W., Feyereisen R., Gonzalez F. J., Coon M. J., Gunsalus I. C., Gotoh O., Okuda K., Nebert D. W. DNA Cell Biol. 12:1-51(1993).[5] Degtyarenko K. N., Archakov A. I. FEBS Lett. 332:1-8(1993).

When tested against Jurket T-cells and U937 cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activation site) promoter element. Thus, it is likely that this gene activates T-cells and myeloid cells through the Jaks-STAT signal transduction pathway. GAS is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

The gene encoding the disclosed cDNA is believed to reside on chromosome 11. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 11.

This gene is expressed primarily in fetal liver spleen, and to a lesser extent in lung, LNCAP prostate cell line, control synovial fibroblasts, human testes tumor, and Hodgkin's lymphoma.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hepatic or developmental diseases and/or disorders, particularly cancer and other proliferative disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the liver, spleen, lung, prostate, testes, and lymphatic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., liver, spleen, developmental, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 69 as residues: Leu-30 to Gly-38, Arg-67 to Val-72, Ser-127 to Trp-133, Gly-148 to Phe-154, Thr-171 to Phe-177, Thr-201 to Asp-206, Ser-265 to Pro-273, Glu-283 to Lys-297, Pro-346 to Lys-357, Phe-409 to Glu-418, Glu-423 to Ser-428, Leu-443 to Cys-448. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in fetal liver and homology to cytochrome P450 monooxygenase indicates indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection and treatment of liver disorders and cancers (e.g., hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells). In addition the expression in fetus would suggest a useful role for the protein product in developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing models and/or tissue trauma. The secreted protein can also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological acitivities. Representative uses are described in the “Chemotaxis” and “Binding Activity” sections below, in Examples 11, 12, 13, 14, 15, 16, 18, 19, and 20, and elsewhere herein. Briefly, the protein may possess the following activities: cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumors); hemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behaviour. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1652 of SEQ ID NO:13, b is an integer of 15 to 1666, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:13, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 4

The translation product of this gene shares sequence homology with C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro (see, e.g., Genbank Accesion number AAB53110.1 (U94333.1); all references available through this accession are hereby incorporated by reference herein. Also see, Immunity 1997 February; 6(2):119-129). Preferred polypeptides encoded by this gene comprise one or more of the following amino acid sequences:

EHPTADRAGCSASGACYSLHHATMKRQAAEEACILRGGALSTVRAGAELRA (SEQ ID NO: 130) VLALLRAGPGPGXGSKDLLFWVALERRRSHCXLENEPLRGFSWLSSDPGGLE SDTLQWVEEPQRSCTARRWV, and/or SRPPVGSSPQLEGDAMPPXRQRYLCKYQFEVLCPAPRPGAASNLSYRAPFQL (SEQ ID NO: 131) HSAALDFSPPGTEVSALCRGQLPISVTCIADEIGARWDKLSGDVLCPCPGRYL HSAALDFSPPGTEVSALCRGQLPISVTCIADEIGARWDKLSGDVLCPCPGRYL RAGKCAELPNCLDDLGGFACECATGFELGKDGRSCVTSGEGQPTLGGTGVPT RRPPATATSPVPQRTWPIRVDEKLGETPLVPEQDNSVTSIPEIPRWGSQSTMST LQMSLQAESKATITPSGSVISKFNSTTSSATPQAFDSSSAVVFIFVSTAVVVLVI LTMTVLGLVKLCFHESPSSQPRKESMGPPGWRVILKPAALGSSSAHCTNNGV KVGDCDLRDRAEGALLAESPLGSSDA.

Polynucleotides encoding such polypeptides are also provided.

This gene is believed to reside on chromosome 14. Therefore, polynucleotides and polypeptides related to this gene are useful in linkage analysis as markers for chromosome 14.

This gene is expressed primarily in chondrosarcoma, smooth muscle tissue, bone marrow, chondrosarcoma, fetal tissue (e.g., heart) and to a lesser extent in ovarian cancer, adult pulmonary tissues, and brain.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, chondrosarcoma, immune disorders, ovarian cancer, respiratory and gastrointestinal disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 70 as residues: Pro-18 to Gly-30. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in bone marrow and homology to C1qR(P) indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer (e.g., ovarian, chondrosarcoma), and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Alternatively, the protein is useful in the detection, treatment, and/or prevention of vascular conditions, which include, but are not limited to, microvascular disease, vascular leak syndrome, aneurysm, stroke, atherosclerosis, arteriosclerosis, or embolism. For example, this gene product may represent a soluble factor produced by smooth muscle that regulates the innervation of organs or regulates the survival of neighboring neurons. Likewise, it is involved in controlling the digestive process, and such actions as peristalsis. Similarly, it is involved in controlling the vasculature in areas where smooth muscle surrounds the endothelium of blood vessels. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2013 of SEQ ID NO:14, b is an integer of 15 to 2027, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:14, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 5

The translation product of this gene shares sequence homology with human renal dipeptidase, which is a glycosyl-phosphatidylinositol-anchored ectoenzyme thought to be important in the metabolism of dihydro peptide bonds (See Genbank Accession No.bbs|148378; dbj|BAA02433.1; gb|AAB59410.1; dbj|BAA02431.1; and Geneseq Accession Nos. W29665 and R30823; all information and references available through these accessions are hereby incorporated herein by reference). Included in this invention as a preferred domain is the renal dipeptidase active site domain, which was identified using the ProSite analysis tool (Swiss Institute of Bioinformatics). Renal dipeptidase (rDP) (EC_(3.4.13.19)), also known as microsomal dipeptidase, is a zinc-dependent metalloenzyme which hydrolyzes a wide range of dipeptides. It is involved in renal metabolism of glutathione and its conjugates. It is a homodimeric disulfide-linked glycoprotein attached to the renal brush border microvilli membrane by a GPI-anchor. A glutamate residue has recently been shown [1] to be important for the catalytic activity of rDP. RDP seems to be evolutionary related to hypothetical proteins in the PQQ biosynthesis operons of Acinetobacter calcoaceticus and Klebsiella pneumoniae. The concensus pattern is as follows: [LIVM]-E-G-[GA]-x(2)-[LIVMF]-x(6)-L-x(3)-Y-x(2)-G-[LIVM]-R [E is the active site residue].

Preferred polypeptides of the invention comprise the following amino acid sequence: VEGGHSLDNSLSILRTFYMLGVR (SEQ ID NO: 137). Polynucleotides encoding these polypeptides are also provided. Further preferred are polypeptides comprising the renal dipeptidase active site domain of the sequence referenced in Table for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence. The additional contiguous amino acid residues may be N-terminal or C-terminal to the renal dipeptidase active site domain. Alternatively, the additional contiguous amino acid residues may be both N-terminal and C-terminal to the renal dipeptidase active site domain, wherein the total N- and C-terminal contiguous amino acid residues equal the specified number. The above preferred polypeptide domain is characteristic of a signature specific to renal dipeptidase proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with renal dipeptidase proteins. Such activities are known in the art, some of which are described elsewhere herein. The following references were referenced above and are hereby incorporated herein by reference: [1] Adachi H., Katayama T., Nakazato H., Tsujimoto M. Biochim. Biophys. Acta 1163:42-48(1993) and [2] Rawlings N. D., Barrett A. J. Meth. Enzymol. 248:183-228(1995). The polypeptide of this gene has been determined to have two transmembrane domains at about amino acid position 17-33 and 470-486 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIb membrane proteins.

When tested against Jurket T-cell cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activation site) and NF-kB (Nuclear Factor kB) pathway. Thus, it is likely that this gene activates T-cells through the Jaks-STAT signal transduction pathway and may be involved in the activation of apoptosis. GAS is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells. Similarly, NF-kB is a transcription factor activated by a wide variety of agents, leading to cell activation, differentiation, or apoptosis. Reporter constructs utilizing the NF-KB promoter element are used to screen supernatants for such activity.

Preferred polypeptides of the invention comprise the following amino acid sequence: RYLTLTH (SEQ ID NO: 132), CNTPWA (SEQ ID NO: 133), APVIFSHS (SEQ ID NO: 134), RNVPDD (SEQ ID NO: 135), GLEDVS (SEQ ID NO: 136), or VEGGHS (SEQ ID NO: 138). Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 16. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 16.

This gene is expressed primarily in infant brain, and to a lesser extent, in primary dendritic cells, L428 cells, melanocytes, keratinocytes, eosinophils, ovarian tumor, thymus stromal cells, treated bone marrow, and Hodgkins lymphoma, and (to a lesser extent) in a variety of other normal and transformed cell types.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, renal, urogenital, or neural disorders, particularly neurodegenerative and/or developmental disorders of the brain, including cancer and other proliferative disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and neurological systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, neural, renal, urogenital, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 71 as residues: Thr-36 to Arg-41, Pro-55 to Pro-60, Pro-67 to Leu-72, Asn-111 to Ser-118, Cys-138 to Asp-144, Asn-290 to Pro-296, Gly-350 to Phe-358, Gly-379 to Glu-384, Gln-399 to Cys-426, Ser-428 to Ser-438. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution combined with its homology to the human renal dipeptidase indicates that this gene or gene product could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilms Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Representative uses are described here and elsewhere herein.

Alternatively, the tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection/treatment of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered bahaviors, including disorders in feeding, sleep patterns, balance, and preception. In addition, the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Moreover, expression within embryonic tissue and other cellular sources marked by proliferating cells combined with the detected GAS and NF-KB biological activity indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2320 of SEQ ID NO:15, b is an integer of 15 to 2334, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:15, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 6

Preferred polypeptides of the invention comprise the following amino acid sequence:

TWLRLGSSQIWLGTAPRGPRIHPEQAGLAGAPVKSTSSEESQPGGQCQ (SEQ ID NO: 139) SSGGAQTLPSLRAAPVAALGSLSSYPDSCPRATTPELCPGAPTLHLADSISGPV SPPGSSLGPDAWTLCAKHHQAKGMTLGTPKVLRLQPVSPCWGPKSWRVPGP FQPGRRRGESRQQGRGKRRSARSAQSPTGPESAAWPC, TVATACVWAACTGCWARPPVPTWAGCAARCAAEDARAGVGDLPATGGAA (SEQ ID NO: 140) TGRRALTPAPPRGPCILSPQPWALGLPGAPLPAALPGRARGRPGLPALPALSTL PGCPALDPAGAGTLCPPPGAAEPAGP, or RSGQPEGSMLRKFSLQRLLSPLDQAQTRWGLALACVAGDKGPPRPWNISSA (SEQ ID NO: 141) PAHPHVTTGMETSGGPARDGGLILEREAAFNKPAPGE.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in immune cells (e.g., eosinophils, T-cells, and macrophage), leukemic and lymphoid cells, rectum, colon, and tonsils.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, inflammatory disorders, particularly immunodeficiency, tumor necrosis, infection, lymphomas, auto-immunities, breast cancer, disorders of the colon and rectum, metastasis, inflammation, anemias (leukemia) and other hematopoeitic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes indicates a usefulness for treatment of cancer (e.g., by boosting immune responses). Expression in cells of lymphoid origin, indicates the natural gene product is involved in immune functions. Therefore it would also be useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Additionally, expression of this gene product in a variety of immune cells indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersentivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

The tissue distribution in kidney indicates the protein product of this gene could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2594 of SEQ ID NO:16, b is an integer of 15 to 2608, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:16, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 7

The translation product of this gene shares sequence homology with the human IgE receptor which is thought to be important in immune regulation, particularly in immune cell aggregation (See Genbank Accession No gi|337418; all references available through this accession are hereby incorporated herein by reference). Moreover, the protein is believed to share structural features to the TM4SF superfamily of proteins. The polypeptide of this gene has been determined to have two transmembrane domains at about amino acid position 73-89 and 106-122 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIa membrane proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with human IgE receptor proteins. Such activities are known in the art, some of which are described elsewhere herein.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

RCQRNKDIMMSSKPTSHAEVNETIPNPYPPSSFMAPGFQQPLGSINLENQAQG (SEQ ID NO: 142) AQRAQPYGITSPGIFASSQPGQGNIQMINPSVGTAVMNFKEEAKALGVIQIMV GLMHIGFGIVLCLISFSFREVLGFASTAXIGGYFWGGLSFIISGSLSVSASKELS RCLVKGSLGMNIGRSILAFIGVILLLVDMCINGVXGQDYWXVLSGKGISATL MIFSXLEFFVACATAHFANQANTTTNMSVLVIPNMYESNPXTPASSSAPPRCN NYSANAPKRKRGISLISWRKTTCKNFLRRCLLLSTMISSL.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in colon.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders and disorders of the digestive tract. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and digestive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, gastrointesinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 73 as residues: Met-2 to Ser-8, Glu-14 to Ser-23, Leu-39 to Gly-53. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution and homology to the human IgE receptor indicates that that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Expression of this gene product in colon indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of proliferative mechanisms in the digestive tract. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene has homology to a gene of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis, and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1277 of SEQ ID NO:17, b is an integer of 15 to 1291, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:17, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 8

This gene is expressed primarily in T-cells and lymph node.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders, particularly immuodeficiencies or inflammatory disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in T-cells and lymph node indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:18 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3115 of SEQ ID NO:18, b is an integer of 15 to 3129, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:18, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 9

The gene encoding the disclosed cDNA is believed to reside on chromosome 2. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 2.

This gene is expressed primarily in immune and haemopoietic cells, particularly messangial cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or haemopoietic diseases. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and haemopoitic systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, haemopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in immune cells and hemopoeitic cells indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3615 of SEQ ID NO:19, b is an integer of 15 to 3629, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:19, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 10

The translation product of this gene shares sequence homology with various dehydrogenase and oxidoreductase polypeptides and appears to belong in the alcohol dehydrogenase Irybitol dehydrogenase family (see, e.g., Genbank accession number AAD36790.1 (AE001811.1) and CAA68181 (X99908.1); all references available through this accession are hereby incorporated by reference herein.) Preferred polypeptides encoded by this gene comprise the following amino acid sequence:

MGRLDGKVIILTAAAQGIGQAAALAFAREGAKVIATDINESKLQELEKYPGIQ (SEQ ID NO: 143) TRVLDVTKKKQIDQFANEVERLDVLFNVAGFVHHGTVLDCEEKDWDFSMNL NVRNVMYLMIKAFLPKMLAQKSGNIINMSSVASSVKGVVNRCVYSTTKAAV IGLTKSVAADFIQQGIRCNCVCPGTVDTPSLQERIQARGNPEEARNDFLKRQK TGRFATAEEIAMLCVYLASDESAYVTGNPVIIDGGWSL.

Also provided are fragments thereof having dehydrogenase activity and polypeptides comprising at least 30 residues of the foregoing amino acid sequence. Polynucleotides encoding such polypeptides are also provided.

This gene is expressed primarily in fetal tissue (e.g., liver, spleen, lung), gall bladder, heart, bone marrow and to a lesser extent in smooth muscle, and parathyroid tumor.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, cardiovascular and developmental disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and fetal systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 76 as residues: Pro-78 to Gln-85, Arg-87 to Arg-94, Asp-96 to Gly-104. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in bone marrow indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1130 of SEQ ID NO:20, b is an integer of 15 to 1144, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 11

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 115-131 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 132 to 152 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

GTIGLLYWVGSIIMSVVVFVPGNIVGKYGTRICPAFFLSIPYTCLPVWAGFRIYN (SEQ ID NO: 144) QPSENYNYPSKVIQEAQAKDLLRRPFDLMLVVCLLLATGFCLFRGLIALDCPS ELCRLYTQFQEPYLKDPAAYPKIQMLAYMFYSVPYFVTALYGLVVPGCSWM PDITLIHAGGLAAQFSHIGASLHARTAYVYRVPEEAKILFLALNIAYGVLP QLLAYRCIYKPEFFIKTAKAEEKVE.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in osteoclastoma, and to a lesser extent, in other human tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, skeletal diseases and/or disorders, particularly osteoclastoma and osteoporosis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., skeletal, immune, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 77 as residues: Thr-32 to Lys-40, Lys-146 to Glu-152. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in osteoclastoma indicates a role in the detection and treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Representative uses are described here and elsewhere herein. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1429 of SEQ ID NO:21, b is an integer of 15 to 1443, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 12

The polypeptide of this gene has been determined to have transmembrane domains at about amino acid position 1-23 and 149-167 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIb membrane proteins.

Preferred polypeptides of the invention comprise the following amino acid sequence:

MSNHDPRGCTRRRAQKPLAIQPRLFHASAPDEGTQKGGCILVQCQ (SEQ ID NO: 145) SEGGAAGAWTGPPSPARDRRVRPPGTKAQRLERRRHVPRLHGLGVGGCEVR TGIVARISGSTPWAGGKPLGLHGAMGEAGAGDTGCCAKGPSPAAPLPAEGRG QGAGPGGLVGRGERRDQQTLLGMAEDTGXPSRPSAPAPRAPVPARQPLPRA RLGAATAISKSRSSRVAPALAAAISASSHQR.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in neutrophils, haemopoietic cells, tymus tumor, osteosarcoma, synovial sarcoma, B-cell lymphoma, dendritic cells, pineal gland, brain, prostate and to a lesser extent in other tissues, including cancers.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and haemopoietic disorders, particularly neutropenia or neutrophilia, and cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and haemopoietic systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, haemopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 78 as residues: Ser-23 to Ala-32, Gly-40 to Glu-47. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in immune cells (e.g., neutrophils) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Expression of this gene product in tonsils also indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

The tissue distribution in pineal gland and brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The tissue distribution in thymus tumor, B-cell lymphoma, osteosarcoma, and synovial sarcoma indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of these and related diseases.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:22 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1039 of SEQ ID NO:22, b is an integer of 15 to 1053, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 13

The translation product of this gene shares sequence homology with lymphoblastic leukemia antigen, which is thought to be important in cancers including leukemia. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 46-62 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 63 to 69 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

This gene is expressed primarily in lung, and infant adrenal gland.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of the pulmonary and endocrine system, including cancers and developmental diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the pulmonary and endocrine systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., pulmonary, endocrine, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in adrenal gland indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of various endocrine disorders and cancers. Representative uses are described in the “Biological Activity”, “Hyperproliferative Disorders”, and “Binding Activity” sections below, in Example 11, 17, 18, 19, 20 and 27, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, and/or prevention of Addison's disease, Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-, hypoparathyroidism), hypothallamus, and testes. Similarly, expression within fetal tissues and other cellular sources marked by proliferating cells, combined with the homology to the human lymphoblastic leukaemia antigen, indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:23 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 727 of SEQ ID NO:23, b is an integer of 15 to 741, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:23, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 14

This gene is expressed primarily in human tonsils.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic diseases and/or disorders, particularly leukemia and cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 80 as residues: Gly-33 to Arg-40, Ser-106 to Met-112, Ala-154 to Gly-163. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in tonsils indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Expression of this gene product indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory conditions such as inflammatory bowel disease, sepsis, acne, and psoriasis.and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 932 of SEQ ID NO:24, b is an integer of 15 to 946, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:24, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 15

The polypeptide of this gene has been determined to have two transmembrane domains at about amino acid position 49-65 and 141-157 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIa membrane proteins.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal pep tide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

STXTXTIGXAGTPAGTGPEFPGRPTRPGEXPVDFSKQYSASWMCLSLLAALA (SEQ ID NO: 146) CSAGDTWASEVGPVLSKSSPRLITTWEKVPVGTNGGVTVVGLVSSLLGGTFV GIAYFLTQLIFVNDLDISAPQWPIIAFGGLAGLLGSIVDSYLGATMQYTGLDES TGMVVNSPTNXARHIAGKPILDNNAVNLFSSVLIALLLPTAAWGFWPRG.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in colon, brain, and to a lesser extent, in epiglottis.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, diseases and/or disorders of the central nervous system and gastrointestinal or digestive tract. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the digestive and central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., gastrointesinal, neural, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection/treatment of neurodegenerative disease states and behavioural disorders. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered bahaviors, including disorders in feeding, sleep patterns, balance, and preception. In addition, the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. The protein product of this gene may also be useful for the detection, treatment, or prevention of a variety of gastrointestinal and digestive tract disorders, particularly proliferative disorders, such as ulcers and cancers. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:25 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 817 of SEQ ID NO:25, b is an integer of 15 to 831, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:25, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 16

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 21-39 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 4041 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins. Preferred polypeptides comprise the following amino acid sequence:

MSQRAGRRPGGWNPSLSVVEVCRGCRGTGPLPWGASLFPCSASPLFPLPLNR (SEQ ID NO: 147) RGDVHGTLGGRMLNRVECRDGVAAAWLCLHDAAAIRGAVGRCPMWTQPT HWVLLLCWALHFYCR

Also preferred are the polynucleotides encoding these polypeptides.

This gene is expressed primarily in tonsils.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders, particularly tonsilitis and tonsillular neoplasms. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, saliva, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Expression of this gene product in tonsils indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersentivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:26 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1280 of SEQ ID NO:26, b is an integer of 15 to 1294, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 17

The gene encoding the disclosed cDNA is believed to reside on chromosome 9. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 9.

This gene is expressed primarily in fetal tissue (e.g., bone, liver, spleen), smooth muscle, chondrosarcoma, osteoblasts, osteosarcoma, and placenta.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of developing and growing organs and tissues, bone disease, osteosarcoma, and other cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the fetal and developing systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., fetal tissue, bone, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, bone marrow, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in osteosarcoma, osteoblasts, and chondrosarcoma indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of bone disease and diseases of the skeletal system. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:27 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1642 of SEQ ID NO:27, b is an integer of 15 to 1656, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:27, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 18

This gene is expressed primarily in liver, fetal liver, and to a lesser extent in bone marrow stromal cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hepatic dysfunction, immune disorders, and disease of the hemopoietic system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the liver and immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, liver, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 84 as residues: Glu-44 to Asp-50. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in liver and fetal liver indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection and treatment of liver disorders and cancers (e.g., hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells). In addition the expression in fetus would suggest a useful role for the protein product in developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing models and/or tissue trauma.

The tissue distribution in bone marrow indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1336 of SEQ ID NO:28, b is an integer of 15 to 1350, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 19

The translation product of this gene shares sequence homology with vacuolar proton-ATPase subunit M9.2 (see, e.g., Genbank accession numbers CAA75571 (Y15286.1); all references available through this accession are hereby incorporated by reference herein.). Preferred polypeptide encoded by this gene comprise the following amino acid sequence:

MTAHSFALPVIIFTTFWGLVGIAGPWFVPKGPNRGVIITMLVATAVCCYLFWL (SEQ ID NO: 148) IAILAQLNPLFGPQLKNETIWYVRFLWE and AQRAARLGTRAPAAPAARPCILPGHPAPGHDGALIRPPGHHLHHVLGPRRHR (SEQ ID NO: 149) GPWFVPKGPNRGVIITML VATAVCCYLFWLIAILAQLNPLFGPQLKNETIWYVRFLWE

Polynucleotides encoding such polypeptides are also provided.

This gene is expressed primarily in infant brain, pancreas islet cell tumor, ovary tumors, immune cells (e.g., T-cells), normal cerebellum, endometrial tumor tissues and to a lesser extent in other tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neurodevelopmental disorders, endocrine system disorders, disorders of the immune system, and ovarian cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the central nervous system, ovaries, immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, neural, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution and homology to vacuolar proton-ATPase subunit M9.2 indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of neurodevelopmental disorders. The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating-diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransrnission, learning, cognition, homeostasis, or neuronal differentiation or survival.

The tissue distribution in immune cells (e.g., T-cells) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The tissue distribution in endocrine tissues such as the pancreas indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of various endocrine disorders and cancers. Representative uses are described in the “Biological Activity”, “Hyperproliferative Disorders”, and “Binding Activity” sections below, in Example 11, 17, 18, 19, 20 and 27, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, and/or prevention of Addison's disease, Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-, hypoparathyroidism), hypothallamus, and testes. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:29 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1752 of SEQ ID NO:29, b is an integer of 15 to 1766, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:29, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 20

This gene is expressed primarily in placenta, induced endothelial cells, immune cells (e.g, T-cells, B-cells, leukocytes), brain, fetal tissue, epididiymus, lung, lung cancer, thyroid tumor and to a lesser extent in many other tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, fetal developmental disorders, immune disorders, cancer of the lungs, thyroid, and cancer, in general. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.

The tissue distribution in immune cells (e.g., T-cells, B-cells, and leukocytes) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation.

The tissue distribution in endothelial cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of disorders involving the vasculature. Elevated expression of this gene product by endothelial cells indicates that it may play vital roles in the regulation of endothelial cell function; secretion; proliferation; or angiogenesis. Alternately, this may represent a gene product expressed by the endothelium and transported to distant sites of action on a variety of target organs. Expression of this gene product by hematopoietic cells also indicates involvement in the proliferation; survival; activation; or differentiation of all blood cell lineages. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:30 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2776 of SEQ ID NO:30, b is an integer of 15 to 2790, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:30, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 21

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 1-29 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins. Preferred polypeptides comprise the following amino acid sequence:

MTLEEHRDRPRLGMCMCVCACVYACMLMHVCVHACLCVCVCVCVEPWSS (SEQ ID NO: 150) R QSKDTGGWHMEEQVTPPSLAQLKSGQVRGEMGEGRGEKGEEALTGGAEA LSLLGRRSPSTPLFLDREDKQ AKDARNLSSTVAPDF

Also preferred are the polynucleotides encoding these polypeptides.

This gene is expressed primarily in tonsils and activated monocytes and to a lesser extent in activated neutrophils and anergic T-cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 87 as residues: Thr-27 to Arg-33, Gly-37 to Ser-42, Pro-52 to Arg-72. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in immunne cells (e.g., T-cells, neutrophils) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Expression of this gene product in tonsils indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:31 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1403 of SEQ ID NO:31, b is an integer of 15 to 1417, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:31, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 22

The gene encoding the disclosed cDNA is believed to reside on chromosome 1. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 1.

This gene is expressed primarily in CD34 positive cells (Cord Blood) and resting T-cells and to a lesser extent in anergic T-cells and neutrophils.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in immune cells (e.g., T-cells, neutrophils) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:32 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1892 of SEQ ID NO:32, b is an integer of 15 to 1906, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:32, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 23

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 33-49 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 50 to 62 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

HEKILTPIWPSSTDLEKPHEMLFLNVILFSLTVFTLISTAHTLDRAVRSDWLLL (SEQ ID NO: 151) VLIYACLEELIPELIF NLYCQGNATLFF.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in activated T cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic diseases and/or disorders, particularly inflammatory conditions or immunodeficiencies such as AIDS. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in T-cells indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:33 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 529 of SEQ ID NO:33, b is an integer of 15 to 543, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 24

This gene is expressed primarily in activated T-cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic diseases and/or disorders, particularly inflammatory or immundeficiency disorders, such as AIDS. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in T-cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Expression of this gene product in indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis, and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:34 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1438 of SEQ ID NO:34, b is an integer of 15 to 1452, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:34, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 25

This gene is expressed primarily in multiple schlerosis tissue, immune cells (e.g., T-cells and dendritic cells), brain, uterus, ovary, stomach, placenta, and fetal tissue.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, multiple sclerosis, disorders of the fetus and female reproductive system, immune disorders, particularly immunodeficiency, tumor necrosis, infection, lymphomas, auto-immunities, cancer, metastasis, wound healing, inflammation, anemias (leukemia) and other hematopoeitic disorders, in addition to developmental or proliferative disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, developmental, reproductive system, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 91 as residues: Met-1 to Lys-6. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in immune cells (e.g., T-cells, dendritic cells) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

The tissue distribution in female reproductive organs indicates that polynucleotides and polypeptides corresponding to this gene are useful for treating female infertility and cancers including but not limited to ovaries and uterus. The protein product is likely involved in preparation of the endometrium of implantation and could be administered either topically or orally. Alternatively, this gene could be transfected in gene-replacement treatments into the cells of the endometrium and the protein products could be produced. Similarly, these treatments could be performed during artificial insemination for the purpose of increasing the likelyhood of implantation and development of a healthy embryo. In both cases this gene or its gene product could be administered at later stages of pregnancy to promote heathy development of the endometrium.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:35 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2894 of SEQ ID NO:35, b is an integer of 15 to 2908, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:35, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 26

The gene encoding the disclosed cDNA is believed to reside on chromosome 20. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 20.

This gene is expressed primarily in fetal and developing tissues, tumors of male and female reproductive tissue (e.g., ovary and testes), and immune cells (e.g., T-cells).

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental and growth disorders, disorders of the immune system, disorders and cancers of ovaries and testes. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the fetal, reproductive, or developing systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developing, differentiating, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 92 as residues: Val-57 to Ala-63. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in immune cells (e.g., T-cells) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The tissue distribution in ovaries and testes indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of diseases of the reproductive organs, including but not limited to ovarian and testicular cancer. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:36 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 939 of SEQ ID NO:36, b is an integer of 15 to 953, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:36, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 27

This gene is expressed primarily in T-cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and haemopoietic disorders, particularly immunodeficiencies, such as AIDS, or inflammatory disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and haemopoietic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, haemopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in T-cells indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3850 of SEQ ID NO:37, b is an integer of 15 to 3864, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:37, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 28

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

PANKAGAAIEAGIGISLMVLSPWACLFVVFFPYIQSSLRSDKHLQLSNILPTPS (SEQ ID NO: 152) HHI HLPASICIQLRAGN.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in brain (early stage human brain and cerebellum) and immune cells (activated neutrophils, activated T-cells, neutrophils and dendritic cells) and to a lesser extent in other tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neural or immune diseases and/or disorders, particularly inflammatory disorders, in addition to cancer and other proliferative disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the brain or the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, neural, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection/treatment of neurodegenerative disease states and behavioural disorders. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered bahaviors, including disorders in feeding, sleep patterns, balance, and preception. In addition, the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system.

Alternatively, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Expression of this gene product in a variety of hematopoietic derived cells (T-cells, neutrophils, etc.) indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis, and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1397 of SEQ ID NO:38, b is an integer of 15 to 1411, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:38, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 29

When tested against Jurket T-cell cell lines, supernatants removed from cells containing this gene activated the NF-kB (Nuclear Factor kB) promoter element. Thus, it is likely that this gene activates T-cells. NF-kB is a transcription factor activated by a wide variety of agents, leading to cell activation, differentiation, or apoptosis. Reporter constructs utilizing the NF-kB promoter element are used to screen supernatants for such activity. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 5-21 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 22 to 40 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

AGSPAGTGPEFPGRPTRPISTHVFEYECICKIPRFMCEYVLLLYIVLLCNRSYA (SEQ ID NO: 153) VFTQCVLRSSPIDSSRNAVLL.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in colon, synovium, chondrosarcoma and to a lesser extent in dendritic cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, gastrointestinal, or skeletal diseases and/or disorders, particularly cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and digestive systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., lymph, skeletal, gastrointestinal, or cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution of this gene product in synovium would suggest a role in the detection and treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). The secreted protein can also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological acitivities, particularly NFk-B activation. Representative uses are described in the “Chemotaxis” and “Binding Activity” sections below, in Examples 11, 12, 13, 14, 15, 16, 18, 19, and 20, and elsewhere herein. Briefly, the protein may possess the following activities: cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumors); hemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behaviour. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:39 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1168 of SEQ ID NO:39, b is an integer of 15 to 1182, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:39, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 30

The gene encoding the disclosed cDNA is believed to reside on chromosome 11. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 11.

This gene is expressed primarily in immune cells (e.g., activated T-cells), testes, fetal, spleen, and to a lesser extent in colon tumor, teratocarcinoma cells, brain and number of other tissues and cell types.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders related to T-cell dysfunction, such as immunodefeciencies or inflammatory conditions, in addition to neural, or reproductive disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, neural, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., seminal fluid, lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 96 as residues: Pro-25 to Arg-38. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in activated T-cells and spleen indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of immune related disorders and diseases, including hypersentivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases; immunodeficiency diseases, such as acquired immunodeficiency syndrome, autoimmunity, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma; infections, and other inflammatory diseases and complications.

Additionally, the tissue distribution in T-cells and spleen indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. In addition, the secreted protein can be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological activities although no evidence for any is provided in the specification. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of haematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation of growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, periodontal disease, neurological diseases stroke, fibrosis); inhibition or stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumours); haemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative disease; for regulation of metabolism, behaviour, and many others. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:40 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2443 of SEQ ID NO:40, b is an integer of 15 to 2457, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:40, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 31

The translation product of this gene shares sequence homology with epidermal growth factor which is thought to be important in the growth and proliferation of epidermal cells, fibroblasts and a variety of other cell types and tissues.

The gene encoding the disclosed cDNA is believed to reside on chromosome 16. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 16.

This gene is expressed primarily in neutrophils.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders, particularly autoimmunities or connective tissue diseases. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and connective tissue systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, epithelial, endothelial, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene are useful for for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1833 of SEQ ID NO:41, b is an integer of 15 to 1847, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:41, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 32

When tested against Reh cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activating sequence) promoter element. Thus, it is likely that this gene activates myeloid cells, and to a lesser extent, other cells and tissue cell types, through the JAK-STAT signal transduction pathway. GAS is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

This gene is expressed primarily in kidneys, tonsils and dendritic cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, renal disorders and neoplasms, tonsilitis and immune disorders, particularly infections. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the renal and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., renal, urogenital, immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in kidney indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of renal conditions such as acute renal failure, kidney fibrosis and kidney tubule regeneration and neoplasms. Conditioned media, generated from the transient expression of this gene in CHO cells has been shown to activate a IFNg-reponsive element (GAS) in a B cell line (Reh). The same conditioned media had no effect on T-cell (Jurkat) and pro-monocyte (U937) derived cell lines, suggesting that the protein product of this gene may exhibit IFNg-like activity in a (B)cell-specific manner. This experimental data in conjunction with expression on dendritic cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g., AIDS), immuno-supressive conditions (transplantation) and hematopoeitic disorders. In addition this gene product may be applicable in conditions of general microbial infection, inflammation or cancer. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ D NO:42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2583 of SEQ ID NO:42, b is an integer of 15 to 2597, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:42, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 33

The translation product of this gene was shown to have homology to the classic MHC transactivator CIITA of Mus musculus (See, e.g., Genbank Accession No gi|1870520 and AAB48859.1; all references available through this accession are hereby incorporated by reference herein.), which is thought to regulate MHC class II gene expression in B lymphocytes via direct interactation with the MHC class II-specific transcription factors. Furthermore, the CIITA protein is thought to play an indirect role in reducing tumorigenicity and inducing long-term tumor immunity.

Preferred polypeptides of the invention comprise the following amino acid sequence:

MPSGNSAAVOUSGKKDKSGBSUSQESAKTKKETKOSCORVREASVBKGSEQSF (SEQ ID NO: 154) RIHFSREDQAGKTLRLSECSFRPEHVSRLATGLSKSLQTELTLTQCCLGQKQL AILLSLVGRPAGLFSLRVQEPWADRARVLSLLEVCAQASGSVTEISISETQQQL CVQLEFPRQEENPEAVALRLAHCDLGAHHSLLXGQLMETCARLXQLSLSQV NLCEDDDASSLLLQSLLLSLSELKTFRLTSSCVSTEGLAHLASGLGHCHHLEEL DLSNNQFDEEGTKALMRALEGKWMLKRLDLSHLLLNSSTLALLTHRLSQMT CLQSLRLNRNSIGDVGCCHLSEALRAATSLEELDLSHNQIGDAGVQHLATILP GLPELRKIDLSGNSISSAGGVQLAESLVLCRRLEELMLGCNALGDPTALGLAQ ELPQHLRVLHLPFSHLGPGGALSLARPWMDPPIWKRSAWRKTTWLEGSCVS VWSSRCSDR, or HQLSRGSAVGRVSRSLQAPGGVDAWLQCPGGSHSPGAGSGAAPAPEGPTPTI (SEQ ID NO: 155) QPSGPRWGPEPGQALDGSPHLEEISLENNLAGGVLRFCMELPLLRQIDLVSC KISNQTAKLLTSSFTSCPALEVILLSWNLLGDEAAAELAQVLPQMGRLKRVD LEKNQITALGAWLLAEGLAQGSSIQVIRLWNNPIPCDMAQHLKSQEPRLDFAF FDNQPQAPWGT.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 16. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 16.

The polypeptide of this gene has been determined to have transmembrane domains at about amino acid positions 1-47 and 24-47 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIa membrane proteins.

This gene is expressed primarily in immune cells (e.g., eosinophils, T-cells, dendritic) and other cell types of hematopoeitic origin and to a lesser extent in ovary tumor and heart.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders, particularly immunodeficiency, tumor necrosis, infection, lymphomas, auto-immunities, cancer, metastasis, inflammation, anemias (leukemia) and other hematopoeitic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in immune cells (T-cells, dendritic, and eosinophils) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:43 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3102 of SEQ ID NO:43, b is an integer of 15 to 3116, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:43, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 34

Polynucleotides of the invention do not comprise the nucleic acid sequence shown as Genbank accession no.gb|Z85986|HS 108K11, which is hereby incorporated herein by reference.

The gene encoding the disclosed cDNA is believed to reside on chromosome 6. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 6.

This gene is expressed primarily in smooth muscle and cells of hematopoeitic origin.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, vascular disorders, particularly heart disease, vasculitis, atherosclerosis, in addition to immune disorders, such as immunodeficiency, auto-immunities, cancer, metastasis, anemias (leukemia) and other hematopoeitic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, endothelial, muscle, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue expression in hematopoeitic tissues indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. In addition, the expression in smooth muscle might indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of cardiovasular and disorders such as atherosclerosis, restenosis, stoke, angina, thrombosis hypertension, inflammation and vascular wound healing. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3446 of SEQ ID NO:44, b is an integer of 15 to 3460, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:44, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 35

The gene encoding the disclosed cDNA is believed to reside on chromosome 17. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 17.

The translation product of this gene was shown to have homology to the human cytochrome c-like polypeptide from lung adenocarcinoma A549 (See Genbank Accession No.bbs|175350) which is thought to be involved in metabolic processes, specific to tumors or transformed cells.

Preferred polypeptides of the invention comprise the following amino acid sequence:

EKLFCFEMLLICKFSPNSVPPETCAILNQGLMDLGLCRMCLGNNMFAGSMLG (SEQ ID NO: 156) KSHRHSPFSINQRHNALRKAAGTPAQKSLGIVQVSPN, GCAGCALVTICLQAVCLVKAIAILHSRLTRDTMHCGRPQGPLPRKAWVLSRF (SEQ ID NO: 157) PPTETA, PETQCTAEGRRDPCPEKPGYCPGFPQLRQPEIWPRGKGKTLHPPARHM, (SEQ ID NO: 158) SEIGENRP, (SEQ ID NO: 159) HDTDSFAH, (SEQ ID NO: 160) ALRKAAG. (SEQ ID NO: 161)

Polynucleotides encoding these polypeptides are also encompassed by the invention. Polynucleotides of the invention do not comprise the nucleic acid sequence shown as Genbank accession no.gb|AC0047061AC004706, which is hereby incorporated herein by reference.

This gene is expressed primarily in fetal tissue, lung, melanocyte, retina, brain, T-cell lymphoma, and to a lesser extent, in other tissues and cell types.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, hepatic, pulmonary, developmental, or growth disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and pulmonary systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, pulmonary, hematopoietic, developing, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.

The tissue distribution in T-cell lymphoma indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Moreover, the expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2608 of SEQ ID NO:45, b is an integer of 15 to 2622, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:45, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 36

The translation product of this gene was shown to have homology to the human protein kinase C substrate 80K-H (See Genbank Accession No. P14314), which is may be important in the regulation of various signal transduction pathways. Polynucleotides of the invention do not comprise the nucleic acid sequence shown as Genbank accession no. gb|G21007|G21007, which is hereby incorporated herein by reference. Additionally, this gene shares sequence homology with a recently described rat neuronal immediate early gene (IEG) cDNA gene R055 (see, e.g., Genseq Accession number Z28293, which is hereby incorporated herein by reference.). An IEG is a gene whose expression is rapidly increased immediately following a stimulus e.g., neuronal stimulation. Such neuronal IEGs have been found to encode a variety of proteins, including transcription factors, cytoskeletal proteins, growth factors and metabolic enzymes, as well as proteins involved in signal transduction. The identification of neuronal IEGs and the proteins they encode may provide important information about the function of neurons in, for example, learning, memory, synaptic transmission, tolerance and neuronal plasticity. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with IEG gene products. Such activities are known in the art, some of which are described elsewhere herein.

This gene is expressed primarily in breast and prostate cancer, retina, ovary, parathyroid tumor, fetal tissue and to a lesser extent in ovary.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive, endocrine, or ocular disorders, particular breast or other cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive and endocrine systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, endocrine, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, breast milk, aquaeous humor, vitreous humor, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 102 as residues: Gln-14 to Val-20, Arg-28 to Trp-35. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in breast cancer cell lines, combined with the homology to a PKC substrate indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, treatment, and/or prevention of a variety of tumors, particularly of the breast or other neoplasms.

Alternatively, the tissue distribution in ovary and parathyroid tumors indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of various endocrine disorders and cancers, particularly Addison's disease, Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-, hypoparathyroidism), hypothallamus, and testes. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The tissue distribution in immune cells (e.g., germinal B-cells) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The tissue distribution in parathyroid tumor, prostate cancer and breast cancer tissue indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment these and related disorders.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1970 of SEQ ID NO:46, b is an integer of 15 to 1984, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 37

Preferred polypeptides of the invention comprise the following amino acid sequence:

MRGPVCGFSLVEMLLALALGLMLILGVTQIALSRTTYASQSAASLLQDDAR (SEQ ID NO: 162) FALGKLIQEIRQAGMFGCLSAASISNAPAGFDRPIGWSTTGSSRSLTLVTADVG EGGSKPDWTVLSDCTGSPPAANARANPLPTCAKLT, or MGYYLSRSRQAGMVLLISLVFLLLLALLGVSSMQGAISQEKITGSLRQRNQSF (SEQ ID NO: 163) QQAESGLRLGESLVQASGFALRPCHSTAACAPPAESVSVVGPGTNPVSTVTWI GMKDGVYGIQNLGPGTGLVNSRQRPRPRSIA.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in cord blood, ovary, tumors of the parathyroid, testes, and pancreas, and to a lesser extent in fetal tissue, retina, brain, colon, endometrial stromal, HL-60 cells, and many other tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, hematopoietic, or developmental disorders, cancer of the ovaries and endocrine system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the hemopoietic and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, reproductive, developmental, endocrine, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 103 as residues: Lys-29 to Ser-38, Ser-55 to Trp-61, Gln-63 to Ser-69. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in endocrine tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of various endocrine disorders and cancers, particularly Addison's disease, Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-, hypoparathyroidism), hypothallamus, and testes.

The tissue distribution in immune cells (e.g., neutrophils and T-cells) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:47 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1973 of SEQ ID NO:47, b is an integer of 15 to 1987, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:47, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 38

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 1-24 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.

This gene is expressed primarily in activated monocytes, dendritic cells, and cancerous ovary.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, ovarian cancer, immune or hematopoietic disorders, particularly immunodeficiencies or inflammatory disordes. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in monocytes and dendritic cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoetic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc.

The tissue distribution in dendritic cells indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The tissue distribution in ovarian cancer tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of ovarian, as well as, other cancers.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:48 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2099 of SEQ ID NO:48, b is an integer of 15 to 2113, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:48, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 39

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 36-57 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type II membrane proteins. Preferred polypeptides encoded by this gene comprise the following amino acid sequence:

ENESTKEPSLLQYLCVQSPAGLNGFNVLLSGSQTPPTVGPSSGQLPSFSVPCM (SEQ ID NO: 164) VLPSPPLGPEFVLYSPAMPGPVSSTLGALPNTGPVNFSLPGLGSIAQLLVGPTA VVNPKSSTLPSADPQLQSQPSLNLSPVMSRSHSVVQQPESPVYVGHPVSVVKL HQSPVPVTPKSIQRTHRETFFKTPGSLGDPVLKRRERNNHETPARPRGD.

Polynucleotides encoding such polypeptides are also provided.

This gene is expressed primarily in 8 week-old embryo, stromal cells, fetal lung, testes, and colon.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive and immune defects, cancer, T-cell lymphoma, and developmental disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the pulmonary and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in T-cell lymphoma indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of developmental and pulmonary defects and neoplasms of blood, reproductive and other organs.

The tissue distribution indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

The tissue distribution in testes tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of male reproductive and endocrine disorders. It may also prove to be valuable in the diagnosis and treatment of testicular cancer, as well as cancers of other tissues where expression has been observed. The expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:49 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3451 of SEQ ID NO:49, b is an integer of 15 to 3465, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:49, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 40

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 145-171 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 168-282 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

This gene is expressed primarily in T helper cells, breast cancer, kidney, fetal tissue and to a lesser extent in thymus and cells from some other hemopoietic and endocrine sources.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and inflammatory conditions, cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the hemopoietic and lymphoid systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 106 as residues: Pro-135 to Ile-145, Trp-173 to Gly-188, Pro-199 to Gln-219, Ser-225 to Ala-237, Pro-240 to Gly-253, Ser-262 to Gly-275. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in breast cancer tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of neoplasms of breast and other organs.

The tissue distribution in immune cells (e.g., T-helper cells) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

The tissue distribution in kidney indicates the protein product of this gene could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Moreover, the expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:50 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1223 of SEQ ID NO:50, b is an integer of 15 to 1237, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:50, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 41

The gene encoding the disclosed cDNA is believed to reside on chromosome 16. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 16.

This gene is expressed primarily in adipose tissue, brain, Hodgkin's lymphoma, and to a lesser extent in fetal tissue colon tumor, synovium, salivary gland, immune cells (e.g., neutrophils), and other tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neurodegenerative disorders, immune, or metabolic disorders, particularly diseases or disorders of adipose tissue or Hodgkin's lymphoma. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the adipose tissues and immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, adipose, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 107 as residues: Arg-54 to Leu-60, Ala-73 to Gly-78. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of obesity, or other metabolic disorders, such as Tay-Sachs disease, phenylkenonuria, galactosemia, hyperlipidemias, porphyrias, and Hurler's syndrome, in addition to various immune disorders and neoplasia.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.

The tissue distribution in immune cells (e.g., neutrophils) indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. In addition, the expression of this gene product in synovium would suggest a role in the detection and treatment of disorders and conditions afflicting the skeletal system, in particular osteoporosis, bone cancer, connective tissue disorders (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation). The protein is also useful in the diagnosis or treatment of various autoimmune disorders (i.e., rheumatoid arthritis, lupus, scleroderma, and dermatomyositis), dwarfism, spinal deformation, joint abnormalities, and chondrodysplasias (i.e. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid, etc.). Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:51 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1383 of SEQ ID NO:51, b is an integer of 15 to 1397, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:51, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 42

Polynucleotides of the invention do not comprise the nucleic acid sequence shown as Genbank accession no.gb|G15452|G|5452, which is hereby incorporated herein by reference.

Preferred polypeptides of the invention comprise the following amino acid sequence:

RHERHEYRRALDHEEEALSSGSVQEAEAMLDEPQEQAEGSLTVYVISEHSSLL (SEQ ID NO: 165) PQDMMSYIGPKRTAVVRGIMHREAFNIIGRRIVQVAQAMSLTEDVLAAALAD HLPEDKWSAEKRRPLKSSLGYEITFSLLNPDPKSHDVYWDIEGAVRRYVQPFL NALGAAGNFSVDSQILYYAMLGVNPRFDSASSSYYLDMHSLPHVINPVESRL GSSAASLYPVLNFLLYVPELAHSPLYIQDKDGAPVATNAFHSPRWGGIMVYN VDSKTYNASVLPVRVEVDMVRVMEVFLAQLRLLFGIAQPQLPPKCLLSGPTS EGLMTWELDRLLWARSVENLATATTTLTSLAQLLGKISNIVIKDDVASEVYK AVAAVQKSAEELASGHLASAFVASQEAVTSSELAFFDPSLLHLLYFPDDQKF AI YIPLFLPMAVPILLSLVKIFLETRKSWRKPEKTD.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in immune and haemopoietic cells, tumors of the ovaries, endometrium, and parathyroid.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or haemopoietic disorders, particularly cancers, ovarian cancer, cancers of the endocrine system and endometrium, and disorders of the retina. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the haemopoietic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, haemopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 108 as residues: Phe-11 to Gly-16, Pro-33 to Ser-42. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in T-cells and bone marrow indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Moreover, the expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:52 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2257 of SEQ ID NO:52, b is an integer of 15 to 2271, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:52, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 43

The gene encoding the disclosed cDNA is believed to reside on chromosome 19. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 19. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 2544 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

This gene is expressed primarily in infant brain, fetal heart, uterine cancer, colon, metastatic melanoma, spleen, liver, thymus and other cancers.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of developing tissues, haemopoietic or immune system, cardiovascular or musculoskeletal, or neural tissues, uterine cancer and metastatic melanoma. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system and developing systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, muscle, immune, hematopoietic, hepatic, developing, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in infant brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection/treatment of neurodegenerative disease states, behavioural disorders, or inflamatory conditions such as Alzheimer's Disease, Parkinson's Disease, Huntingtons Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered bahaviors, including disorders in feeding, sleep patterns, balance, and preception. In addition, the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Alternatively, expression within embryonic tissue and other cellular sources marked by proliferating cells indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. The secreted protein can also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological acitivities. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumors); hemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behaviour. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures.

The tissue distribution in uterine cancer and metastatic melanoma indicates that polynucleotides and polypeptides corresponding to this gene are useful for the detection/treatment of these cancers.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:53 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2755 of SEQ ID NO:53, b is an integer of 15 to 2769, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:53, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 44

This gene shares sequence homology with Serglycin (see GenSeq accession number Q44278; all references available through this accession are hereby incorporated by reference herein.) Serglycin is involved in the regulation of haematopoietic cell function and development.

This gene is expressed primarily in brain frontal cortex.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neurological and cognitive conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the central nervous system (CNS), expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 110 as residues: Tyr-28 to Cys-40. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of central nervous system disorders, esp. schizophrenia, neurodegenerative and memory disorders.

The tissue distribution in brain indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:54 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1375 of SEQ ID NO:54, b is an integer of 15 to 1389, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:54, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 45

This gene is expressed primarily in human stomach.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal disorders, particularly gastritis, stomach ulcers, and stomach cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the gastrointestinal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., gastrointesinal, endothelial, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in stomach cells and tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for treating or diagnosing disease involving the stomach such as inflammation, ulceration or cancers. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:55 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 734 of SEQ ID NO:55, b is an integer of 15 to 748, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:55, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 46

The gene encoding the disclosed cDNA is believed to reside on chromosome 11. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 11.

This gene is expressed primarily in brain and fetal liver, and to a lesser extent, in other cell types.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental, neurological, behavioral, hepatic or immune diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune or nervous systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., hepatic, immune, hematopoietic, neural, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 112 as residues: Ala-24 to Lys-31. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution of this gene predominantly in fetal liver indicates that the gene could be important for the treatment or detection of immune or hematopoietic disorders including arthritis, asthma, immunodeficiency diseases and leukemia. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder and panic disorder. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:56 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 4188 of SEQ ID NO:56, b is an integer of 15 to 4202, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:56, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 47

Preferred polypeptides of the invention comprise the following amino acid sequence:

KLLLTKVEQKLELARLQVDTSGSKEFGTSGIPAKCRFPKIFVNTDDTYEELHLI (SEQ ID NO: 166) VYKVTTVFLPAL.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in brain and lymph node of breast cancer.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, breast cancer and neural disorders, particular neurodegenerative, neurological, or psycholigical disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of neurological and psychological disorders, including but not limited to: trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, toxic neuropathies induced by neurotoxins, inflammatory diseases such as meningitis and encephalitis, demyelinating diseases, neurodegenrative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, peripheral neuropathies, multiple sclerosis, neoplasia of neuroectodermal origin, etc. In addition, the secreted protein can be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological activities although no evidence for any is provided in the specification. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of haematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation of growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, periodontal disease, neurological diseases stroke, fibrosis); inhibition or stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumours); haemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative disease; for regulation of metabolism, behaviour, and many others. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures.

The tissue distribution in cancerous breast tissue indicates polynucleotides and polypeptides corresponding to this gene are useful for the detection, treatment, and/or prevention of breast cancer, and cancer in general. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:57 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 840 of SEQ ID NO:57, b is an integer of 15 to 854, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:57, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 48

A preferred polypeptide fragment of the invention comprises the following amino acid sequence:

MEPQLGPEAAALRPGWLALLLWVSALSCSFSLPASSLSSLVPQVRTSYNFGRT (SEQ ID NO: 167) FLGLDKCNACIGTSICKKFFKERNKI.

Polynucleotides encoding these polypeptides are also provided.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

QLPLWPSPASVQPRVDSQRARGSPEPKMEPQLGPEAAALRPGWLALLLWVS (SEQ ID NO: 168) ALSCSFSLPASSLSSLVPQVRTSYNFGRTFLGLDKCNACIGTSICKKFFKEEIRS DNWLASHLGLPPDSLLSYPANYSDDSKIWRPVEIFRLVSKYQNEISDRKICAS ASAPKTCSIERVLRKTERFQKWLQAKRLTPDLVQDC HQCQRELKFLCMLR.

Polynucleotides encoding these polypeptides are also provided.

A preferred polypeptide variant of the invention comprises the following amino acid sequence:

MEPQLGPEAAALRPGWLALLLWVSALSCSFSLPASSLSSLVPQVRTSYNFGRT (SEQ ID NO: 169) FLGLDKCNACIGTSICKKFFKEEIRSDNWLASHLGTASRFPLXSYPCKLLQMIX KIWXPCGXLLTGQQXSNEISKQEIXCLLHPPPKNLHIDV.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in endothelial cells, and to a lesser extent, in the adult pulmonary system.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, vascular and pulmonary diseases and/or disorders, particularly atherosclerosis, and microvascular disease. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the pulmonary and vascular systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endothelial, pulmonary, cardiovascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, pulmonary surfactant, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 114 as residues: Arg-45 to Gly-51, Glu-75 to Asn-81, Ala-99 to Ile-107, Lys-119 to Asp-126, Leu-145 to Gln-152. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution of this gene in the pulmonary system, and in particular endothelial cells, indicates that it could play a role in the treatment/detection of lung lymphoma or sarcoma formation, pulmonary edema and embolism, bronchitis and cystic fibrosis. Expression in endothelial cells suggest a role in the treatment and/or detection of vascular disorders including vasculitis, cardiovascular disorders such as myocardial infarction, myocarditis, ischemia and stroke. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:58 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1441 of SEQ ID NO:58, b is an integer of 15 to 1455, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:58, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 49

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence:

GPRARVQGFSGADIVKFMALGSMYLVLTLIVAKVLRGAEPCCGPLKNRVLRP (SEQ ID NO: 170) CPLPVHCPLPIPSPAEGIPWVAYLPIRWFISCCPGHCIQIPMCTS.

Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in kidney, and to a lesser extent, in a wide variety of human tissues.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, renal or urogenital diseases and/or disorders, particularly kidney cancer or nephritis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the urological or renal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., renal, urogenital, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in kidney indicates that this gene or gene product could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilms Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Representative uses are described here and elsewhere herein. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:59 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 579 of SEQ ID NO:59, b is an integer of 15 to 593, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:59, and where b is greater than or equal to a+14.

TABLE 1 5′ NT First Last ATCC NT 5′ NT 3′ NT 5′ NT of First AA AA AA First Last Deposit SEQ Total of of of AA of SEQ of of AA of AA Gene cDNA No: Z ID NT Clone Clone Start Signal ID Sig Sig Secreted of No. Clone ID and Date Vector NO: X Seq. Seq. Seq. Codon Pep NO: Y Pep Pep Portion ORF 1 HKGCN17 203105 pSport1 11 3289 1 3289 218 218 67 1 23 24 155 08/13/98 2 HETAD68 203081 Uni-ZAP XR 12 2342 1 2342 84 84 68 1 32 33 69 07/30/98 3 HPIAT78 203081 Uni-ZAP XR 13 1666 1 1666 30 30 69 1 29 30 501 07/30/98 4 HMWGY65 203105 Uni-ZAP XR 14 2027 1 1976 42 42 70 1 21 22 188 08/13/98 5 HDTAB58 203081 pCMVSport 15 2334 1874 2334 133 133 71 1 33 34 486 07/30/98 2.0 5 HDTAB58 203081 pCMVSport 60 496 159 496 223 223 116 1 37 38 48 07/30/98 2.0 6 HEOMQ62 203105 pSport1 16 2608 1 2608 130 130 72 1 24 25 87 08/13/98 7 HWLJQ88 203081 pSport1 17 1291 1 1291 114 114 73 1 30 31 299 07/30/98 7 HWLJQ88 203081 pSport1 61 1292 1 1292 403 403 117 1 25 26 203 07/30/98 8 HMICP03 203105 Uni-ZAP XR 18 3129 1 3129 86 86 74 1 34 35 47 08/13/98 9 HAJAB01 203105 pCMVSport 19 3629 1 3629 147 147 75 1 15 16 43 08/13/98 3.0 10 HE2AT09 203105 Uni-ZAP XR 20 1144 437 1144 435 435 76 1 17 18 140 08/13/98 11 HSDJA15 203081 Uni-ZAP XR 21 1443 1 1443 247 247 77 1 20 21 152 07/30/98 12 HAMGW29 203105 pCMVSport 22 1053 12 1053 25 25 78 1 27 28 179 08/13/98 3.0 13 HAPSR85 203081 Uni-ZAP XR 23 741 1 741 428 428 79 1 21 22 69 07/30/98 14 HTOHD42 203081 Uni-ZAP XR 24 946 1 946 155 155 80 1 24 25 190 07/30/98 15 HWLIH65 203081 pSport1 25 831 1 831 129 129 81 1 18 19 165 07/30/98 16 HTOJA73 203105 Uni-ZAP XR 26 1294 1 1294 100 100 82 1 21 22 41 08/13/98 17 HPMGJ45 203105 Uni-ZAP XR 27 1656 1 1656 119 119 83 1 25 26 48 08/13/98 18 HFVIC62 203105 pBluescript 28 1350 1 1350 114 114 84 1 31 32 56 08/13/98 19 HHENW77 203105 pCMVSport 29 1766 1 1714 205 205 85 1 19 20 42 08/13/98 3.0 20 HMSIV91 203105 Uni-ZAP XR 30 2790 1 2790 95 95 86 1 19 20 40 08/13/98 21 HMSKC04 203105 Uni-ZAP XR 31 1417 1 1417 133 133 87 1 22 23 73 08/13/98 22 HSAZG33 203105 Uni-ZAP XR 32 1906 1 1906 122 122 88 1 23 24 46 08/13/98 23 HTEBC92 203081 Uni-ZAP XR 33 543 1 543 63 63 89 1 22 23 62 07/30/98 23 HTEBC92 209215 Uni-ZAP XR 62 398 241 398 156 118 1 13 14 18 08/21/97 24 HTXEL29 203081 Uni-ZAP XR 34 1452 1 1452 322 322 90 1 25 26 69 07/30/98 24 HTXEL29 209090 Uni-ZAP XR 63 1202 1 1202 294 119 1 12 06/05/97 25 HDPAW44 203105 pCMVSport 35 2908 1 2908 40 40 91 1 30 31 56 08/13/98 3.0 26 HMACS20 203105 Uni-ZAP XR 36 953 1 953 227 227 92 1 43 44 67 08/13/98 27 HAJAY88 203105 pCMVSport 37 3864 1 3864 201 201 93 1 25 26 44 08/13/98 3.0 28 HBOEG69 203081 pSport1 38 1411 1 1411 302 302 94 1 19 20 54 07/30/98 29 HWLEQ37 203081 pSport1 39 1182 1 1182 110 110 95 1 19 20 40 07/30/98 30 HE9CS37 203105 Uni-ZAP XR 40 2457 65 2454 183 183 96 1 22 23 40 08/13/98 31 HNGEI34 203105 Uni-ZAP XR 41 1847 1 1847 70 70 97 1 18 19 49 08/13/98 32 HTOAT76 203105 Uni-ZAP XR 42 2597 1 2597 100 100 98 1 41 42 57 08/13/98 33 HDPVH60 203105 pCMVSport 43 3116 1 3100 8 8 99 1 45 46 51 08/13/98 3.0 34 HLYCR65 203105 pSport1 44 3460 1 3460 28 28 100 1 19 20 44 08/13/98 35 HARAY91 203105 pBluescript 45 2622 1 2622 214 214 101 1 46 47 57 08/13/98 SK- 36 HCHNT03 203105 pSport1 46 1984 133 1984 228 228 102 1 17 18 40 08/13/98 37 HCUBW95 203105 ZAP Express 47 1987 1 1987 131 131 103 1 20 21 142 08/13/98 38 HDPLV95 203105 pCMVSport 48 2113 1 2113 12 12 104 1 18 19 43 08/13/98 3.0 39 HEMGB12 203105 Uni-ZAP XR 49 3465 1 3438 78 78 105 1 48 49 62 08/13/98 40 HHENP27 203105 pCMVSport 50 1237 1 1237 12 12 106 1 22 23 282 08/13/98 3.0 41 HSPBF70 203105 pSport1 51 1397 288 1397 429 429 107 1 19 20 97 08/13/98 42 HTXKB57 203105 Uni-ZAP XR 52 2271 1 2271 290 290 108 1 16 17 61 08/13/98 43 HUKAA55 203105 Lambda ZAP 53 2769 129 2769 263 263 109 1 32 33 46 08/13/98 II 44 HFXGT58 203105 Lambda ZAP 54 1389 12 1389 238 238 110 1 28 29 46 08/13/98 II 45 HROAS46 203081 Uni-ZAP XR 55 748 1 748 123 123 111 1 19 20 41 07/30/98 46 HUSFF19 203081 pBluescript 56 4202 863 2447 1080 1080 112 1 20 21 41 07/30/98 47 HBWBX21 203105 ZAP Express 57 854 1 854 201 201 113 1 17 18 43 08/13/98 48 HUVDJ43 203081 Uni-ZAP XR 58 1455 103 1455 128 128 114 1 31 32 182 07/30/98 48 HUVDJ43 203081 Uni-ZAP XR 64 1517 1 1517 133 133 120 1 31 32 79 07/30/98 48 HUVDJ43 203027 Uni-ZAP XR 65 526 69 526 89 89 121 1 31 32 146 06/26/97 49 HTLCU49 203081 Uni-ZAP XR 59 593 1 593 170 170 115 1 20 21 80 07/30/98 49 HTLCU49 203027 Uni-ZAP XR 66 664 1 664 249 249 122 1 20 21 80 06/26/97

Table 1 summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table 1 and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” Some of the deposits contain multiple different clones corresponding to the same gene. “Vector” refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq.” refers to the total number of nucleotides in the contig identified by “Gene No.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.”

The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” The predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion.” Finally, the amino acid position of SEQ ID NO:Y of the last amino acid in the open reading frame is identified as “Last AA of ORF.”

SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:3140 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA contained in ATCC deposit Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.

Signal Sequences

The present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues (i.e., + or −5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as desribed below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.

The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence contained in a deposited cDNA clone or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in a deposited clone, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).

By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown in Table 1, the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, an amino acid sequences shown in Table 1 (SEQ ID NO:Y) or to the amino acid sequence encoded by cDNA contained in a deposited clone can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” (See, Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments of the polynucleotides of the invention.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.

The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the invention for binding to an antibody of the polypeptide of the invention, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In another embodiment, where a ligand for a polypeptide of the invention identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123. In another embodiment, physiological correlates of binding of a polypeptide of the invention to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.

Epitopes and Antibodies

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666(1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA-segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 1×10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferrably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2): 177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples (e.g., Example 16). In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92101047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (ANIC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:73749 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion-assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114(1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O₂. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O₂. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., NY, and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NABH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide seuqence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.

In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the present invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the present invention, where each probe has one strand containing a 31′mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.

Where a diagnosis of a disorder, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the present invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.

By “measuring the expression level of polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including cancerous diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.

Pathological cell proliferative diseases, disorders, and/or conditions are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO 91/15580) However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5′ end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness would not be limited to treatment of proliferative diseases, disorders, and/or conditions of hematopoietic cells and tissues, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.

Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods for treatingor preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the nakednucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is herein incorporated by reference), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology, 101:512-527 (1983), which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca²⁺-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 9419469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CAPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)).

A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Determining an effective amount of substance, to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer or some autoimmune diseases, disorders, and/or conditions, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies), blood platelet diseases, disorders, and/or conditions (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful in treating, preventing, and/or diagnosing autoimmune diseases, disorders, and/or conditions. Many autoimmune diseases, disorders, and/or conditions result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune diseases, disorders, and/or conditions.

Examples of autoimmune diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat, prevent, and/or diagnose hyperproliferative diseases, disorders, and/or conditions, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative diseases, disorders, and/or conditions can be treated, prevented, and/or diagnosed. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating, preventing, and/or diagnosing hyperproliferative diseases, disorders, and/or conditions, such as a chemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative diseases, disorders, and/or conditions can also be treated, prevented, and/or diagnosed by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders, and/or conditions include, but are not limited to: hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or conditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventing cell proliferative diseases, disorders, and/or conditions by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method of treating or preventing cell-proliferative diseases, disorders, and/or conditions in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the poynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferrably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating, preventing, and/or diagnosing one or more of the described diseases, disorders, and/or conditions. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the present invention are useful for treating, preventing, and/or diagnosing a subject having or developing cell proliferative and/or differentiation diseases, disorders, and/or conditions as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases, disorders, and/or conditions related to polynucleotides or polypeptides, including fragements thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragements thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses. 50(5):423-33 (1998), Chem Biol Interact. Apr 24;111-12:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int J Tissue React; 20(1):3-15 (1998), which are all hereby incorporated by reference).

Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat, prevent, and/or diagnose cardiovascular diseases, disorders, and/or conditions, including peripheral artery disease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders, and/or conditions, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye diseases, disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases, disorders, and/or conditions associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating, preventing, and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treator prevent a cancer or tumor. Cancers which may be treated, prevented, and/or diagnosed with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat or prevent cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating, preventing, and/or diagnosing other diseases, disorders, and/or conditions, besides cancers, which involve angiogenesis. These diseases, disorders, and/or conditions include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods are provided for treating, preventing, and/or diagnosing hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating, preventing, and/or diagnosing neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular diseases, disorders, and/or conditions associated with neovascularization which can be treated, prevented, and/or diagnosed with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are provided for treating or preventing neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of diseases, disorders, and/or conditions can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical bums). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating or preventing neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat or prevent early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating or preventing proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided for treating or preventing retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

Additionally, diseases, disorders, and/or conditions which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, diseases, disorders, and/or conditions and/or states, which can be treated, prevented, and/or diagnosed with the the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha, alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be treated, prevented or diagnosed by the polynucleotides or polypeptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative diseases, disorders, and/or conditions (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associted with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment subsequent to dermal loss.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote skin strength and to improve the appearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated, prevented, and/or diagnosed using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can be treated, prevented, and/or diagnosed with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases, disorders, and/or conditions which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated, prevented, and/or diagnosed in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases, disorders, and/or conditions, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral hypoxia. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral ischemia. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral infarction. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose or prevent neural cell injury associated with a stroke. In a further aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/or conditions that may be treated, prevented, and/or diagnosed according to the invention include, but are not limited to, diseases, disorders, and/or conditions such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as diseases, disorders, and/or conditions that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat, prevent, and/or diagnose infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated, prevented, and/or diagnosed. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picomaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Kiebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used totreat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, bums, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated, prevented, and/or diagnosed include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated, prevented, and/or diagnosed using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic diseases, disorders, and/or conditions (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated, prevented, and/or diagnosed using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention.

Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat, prevent, and/or diagnose inflammation, infection, hyperproliferative diseases, disorders, and/or conditions, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat, prevent, and/or diagnose wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat, prevent, and/or diagnose wounds.

It is also contemplated that a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used totreat, prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labelled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides of the invention may be alterred by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat, prevent, and/or diagnose disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptidelmolecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with the polypeptide, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.

As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84103564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/HindIII site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA corresponding to the polynucleotides of the invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.

Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.

invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention

Other Activities

The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due to injuries, bums, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.

Similarly preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.

A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X.

Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

Also preferred is a composition of matter comprising a DNA molecule which comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table 1 for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone identified by a cDNA Clone Identifier in Table 1, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table 1.

Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.

Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1.

Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:Y in the range of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a secreted portion of the secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in able 1.

Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1 and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y is defined in Table 1; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

In specific embodiments of the invention, for each “Contig ID” listed in the fourth column of Table 2, preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, a nucleotide sequence referenced in the fifth column of Table 2 and described by the general formula of a−b, whereas a and b are uniquely determined for the corresponding SEQ ID NO:X referred to in column 3 of Table 2. Further specific embodiments are directed to polynucleotide sequences excluding one, two, three, four, or more of the specific polynucleotide sequences referred to in the fifth column of Table 2. In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.

TABLE 2 NT SEQ ID cDNA Clone NO: Gene No. ID X Contig ID Public Accession Numbers  1 HKGCN17 11 798098 None  2 HETAD68 12 793860 R74475, H18234, H18272, H25658, H26245, N33532, N41513, N93486, W02757, W16473, W16474, W21189, W24767  3 HPIAT78 13 790205 T91507, T91536, R11612, T83549, T83719, R19402, AA058765, AA088847, AA099882, AA115449, AA115448, AA235247  4 HMWGY65 14 794987 R14788, R40100, R51562, R51653, H46612, AA004670  5 HDTAB58 15 800678 R13499, R18648, R20620, R41610, R41610, R78726, H10829, H10870  5 HDTAB58 60 793685 R44548, R44548, H42753, H98068, N24692, N32182, AA463629 10 HE2AT09 20 710408 T58287, T58241, R51009, H47150, H66260, H69720, H70386, H70698, H72155, H91380, N38922, N46494, N58822, N72645, N75027, N75160, N93947, N94250, W05627, W07723, W19532, W39201, W40446, W80561, N90260, AA022870, AA026818, AA026877, AA036917, AA055012, AA055436, AA057027, AA136969, AA460271, AA463838 11 HSDJA15 21 795252 T87220, R41971, R45396, R41971, R45396, R55771, H11649 12 HAMGW29 22 799448 H44706, W69343, AA085550, AA148240 19 HHENW77 29 797475 T74074, T77194, T87227, R39444, H43568 23 HTEBC92 62 495967 T78329, R70701 25 HDPAW44 35 790193 R27595, P27693, N39440, N48553, AA036790, AA036733, AA169377, AA186377, AA233190 28 HBOEG69 38 793786 R27740, R95913, H80936, W74158 30 HE9CS37 40 797496 R34404, R48942, H17123, H17230, H25265, H41988, H41987, N20900, AA040195, AA128254, AA256865, AA255468, AA461458, AA426036 33 HDPVH60 43 796865 T61473, AA005022, AA005023, AA227825, AA227999 36 HCHNT03 46 797709 H87265, W19728, N89985, AA459996, AA460090 41 HSPBF70 51 793744 R39978, R39978, AA128437, AA129952, AA136304, AA136410 46 HUSFF19 56 797713 T60523, T61826, T64314, T64701, T64884, T89070, T98251, R18180, R25532, R25699, R34919, R45591, R45670, R49311, R49311, R45670, R62578, R62579, R66150, R67772, H17920, H27973, H29126, H40149, H40159, H54660, H54661, H66579, H70677, H80692, H80693, H94982, W02716, W21368, W25735, W67720, W68179, W72502, W76059, AA004905, AA004999, AA009733, AA009449, AA034233, AA043665, AA043790, AA053925, AA134987, AA135085, AA220761, AA213510, AA214529 49 HTLCU49 66 695847 N51554, AA057753, AA458613

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLES Example 1

Isolation of a Selected cDNA Clone from the Deposited Sample

Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector. Table 1 identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The table immediately below correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 1 as being isolated in the vector “Lambda Zap,” the corresponding deposited clone is in “pBluescript.”

Vector Used to Construct Library Plasmid Corresponding Deposited Lambda Zap pBluescript (pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ®2.1 pCR ®2.1

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK−, KS+and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S” is for SacI and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the fl origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the fl ori generates sense strand DNA and in the other, antisense.

Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).) Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 1, as well as the corresponding plasmid vector sequences designated above.

The deposited material in the sample assigned the ATCC Deposit Number cited in Table 1 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table 1. Typically, each ATCC deposit sample cited in Table 1 comprises a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; but such a deposit sample may include plasmids for more or less than 50 cDNA clones, up to about 500 cDNA clones.

Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNAs cited for that clone in Table 1. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 3040 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with ³²P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5′ NT and the 3′ NT of the clone defined in Table 1) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.

This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.

Example 2

Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO:X., according to the method described in Example 1. (See also, Sambrook.)

Example 3

Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 1 is labeled with P³² using the rediprime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (Clontech) are examined with the labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70 degree C. overnight, and the films developed according to standard procedures.

Example 4

Chromosomal Mapping, of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5

Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan^(r)). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.

In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operator sequences are made synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.

Example 6

Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.

The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.

To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀ monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 7

Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).

Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified in Table 1, is amplified using the PCR protocol described in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

Five ug of a plasmid containing the polynucleotide is co-transfected with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of ³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 8

Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC₃₇₁₅₂), pSV2dhfr (ATCC₃₇₁₄₆), pBC12MI (ATCC₆₇₁₀₉), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CVI, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.

A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five μg of the expression plasmid pC6 a pC4 is cotransfected with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 9

Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)

Human IgG Fc Region:

GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC (SEQ ID NO: 1) CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGT GGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10

Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the secreted protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

Example 11

Production of Secreted Protein for High-Throughput Screening Assays

The following protocol produces a supernatant containing a polypeptide to be tested. This supernatant can then be used in the Screening Assays described in Examples 13-20.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in 0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604 F Biowhittaker))/10% heat inactivated FBS(14-503 F Biowhittaker)/1× Penstrep(17-602 E Biowhittaker). Let the cells grow overnight.

The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8 or 9, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 1545 minutes. After about 20 minutes, use a multi-channel pipetter to add 150 ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a 12-channel pipetter with tips on every other channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degrees C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1%BSA in DMEM with 1×penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L of FeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L of MgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L of NaH₂PO₄-H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002 mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal) with 2 mm glutamine and lx penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each well. Incubate at 37 degrees C. for 45 or 72 hours depending on the media used: 1%BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 13-20.

It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide directly (e.g., as a secreted protein) or by the polypeptide inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.

Example 12

Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with L-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID NO:2)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway.

Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.

JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISRE IFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1>Lys6 >IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotrophic) + + + ? 1, 3 GAS (IRF1>Lys6>IFP) Il-11 (Pleiotrophic) ? + ? ? 1, 3 OnM (Pleiotrophic) ? + + ? 1, 3 LIF (Pleiotrophic) ? + + ? 1, 3 CNTF (Pleiotrophic) −/+ + + ? 1, 3 G-CSF (Pleiotrophic) ? + ? ? 1, 3 IL-12 (Pleiotrophic) + − + + 1, 3 g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − + − + 6 GAS (IRF1=IFP>>Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1>IFP>>Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growth hormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS(B-CAS>IRFI=IFP>>Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 13-14, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is:

5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC (SEQ ID NO: 3) GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site:

5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)

PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:

5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA (SEQ ID NO: 5) TGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCG CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT CCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC TCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT AGGCTTTTGCAAAAAGCTT:3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14.

Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 15 and 16. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 13

High-Throughput Screening Assay for T-Cell Activity.

The following protocol is used to assess T-cell activity by identifying factors, and determining whether supernate containing a polypeptide of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1%Pen-Strep. Combine 2.5 mis of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down the required number of cells (10⁷ per transfection), and resuspend in OPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of 1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degrees C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptides of the invention and/or induced polypeptides of the invention as produced by the protocol described in Example 11.

On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at −20 degrees C. until SEAP assays are performed according to Example 17. The plates containing the remaining treated cells are placed at 4 degrees C. and serve as a source of material for repeating the assay on a specific well if desired.

As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.

The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.

Example 14

High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity by determining whether polypeptides of the invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10⁷ U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂. Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degrees C. for 36 hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×10⁵ cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described in Example 11. Incubate at 37 degrees C. for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 17.

Example 15

High-Throughput Screening Assay Identifying Neuronal Activity.

When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed.

Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells can be assessed.

The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:

(SEQ ID NO: 6) 5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO: 7) 5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′

Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamine protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced by Example 11, 37° C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 17.

Example 16

High-Throughput Screening Assay for T-Cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 11. Activators or inhibitors of NF-KB would be useful in treating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site:

5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGACTTTCCGGGAC (SEQ ID NO: 9) TTTCCATCCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site:

5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)

PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:

5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC (SEQ ID NO: 10) ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGA CTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTA TTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT:3′

Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 13. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 13. As a positive control, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.

Example 17

Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of 2.5× dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on luminometer, one should treat 5 plates at each time and start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.

Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD (ml) 10  60 3 11  65 3.25 12  70 3.5 13  75 3.75 14  80 4 15  85 4.25 16  90 4.5 17  95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 18

High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO₂ incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The supernatant is added to the well, and a change in fluorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event which has resulted in an increase in the intracellular Ca⁺⁺ concentration.

Example 19

High-Throughput Screening Assay Identifying Tyrosine Kinase Activity

The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulating tyrosine kinase activity, the identification of novel human secreted proteins capable of activating tyrosine kinase signal transduction pathways are of interest. Therefore, the following protocol is designed to identify those novel human secreted proteins capable of activating the tyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example 11, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4 degrees C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degrees C. at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of 5 uM Biotinylated Peptide, then 10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl₂, 5 MM MnCl₂, 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate (1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30 degrees C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degrees C. for 20 min. This allows the streptavadin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase (anti-P-Tyr-POD (0.5 u/ml)) to each well and incubate at 37 degrees C. for one hour. Wash the well as above.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.

Example 20

High-Throughput Screening Assay Identifying Phosphorylation Activity

As a potential alternative and/or compliment to the assay of protein tyrosine kinase activity described in Example 19, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degrees C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 11 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.

After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/mil) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation.

Example 21

Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 22

Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 23

Formulation

The invention also provides methods of treatment and/or prevention diseases, disorders, and/or conditions (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

Therapeutics can be are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLET™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/ANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™, IVEEGM™, SANDOGLOBULIN™, GAMMAGARD S/D™, and GAMIMUNE™. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In another embodiment, compostions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estrarnustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP. In another embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a further embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL11, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PDGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE™ (SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 24

Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 23.

Example 25

Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided in Example 23.

Example 26

Method of Treatment Using Gene Therapy-ex vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 27

Gene Therapy Using Endogenous Genes Corresponding to Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×10⁶ cells/ml. Electroporation should be performed immediately following resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC₁₈ plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters; a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 28

Method of Treatment Using Gene Therapy—in vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(l):1-7) which can be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 29

Transgenic Animals.

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 30

Knock-Out Animals.

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 31

Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing XXX are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of XXX protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

Monoclonal antibodies specific for protein XXX are prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with XXX polypeptide or, more preferably, with a secreted XXX polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the XXX polypeptide.

Alternatively, additional antibodies capable of binding to XXX polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the XXX protein-specific antibody can be blocked by XXX. Such antibodies comprise anti-idiotypic antibodies to the XXX protein-specific antibody and are used to immunize an animal to induce formation of further XXX protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

b) Isolation of Antibody Fragments Directed

Against XXX from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against XXX to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13 delta gene m, see PCT publication WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/mil ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/mil of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.

Example 32

Assays Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation

Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.

In vitro Assay

Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.

In vivo Assay

BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220) dull B cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 33

T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of ³H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×10⁴ well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of polypeptides of the invention (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatant is removed and stored −20 degrees C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of ³H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative controls for the effects of polypeptides of the invention.

The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 34

Effect of Polypeptides of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the Production of Cytokines.

Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Th1 helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (10⁶/ml) are treated with increasing concentrations of polypeptides of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e.g, R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte Activation and/or Increased Survival.

Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Polypeptides, agonists, or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay.

Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated process (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubaed at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.

Effect on Cytokine Release.

An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×10⁵ cells/mil with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g, R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.

Oxidative Burst.

Purified monocytes are plated in 96-w plate at 2-1×10⁵ cell/well. Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37° C. for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H₂O₂ produced by the macrophages, a standard curve of a H₂O₂ solution of known molarity is performed for each experiment.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polypeptides, polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 35

Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays

Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE₂ assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-1α for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released. Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP⁺ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 36

The Effect of Polypeptides of the Invention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO:Y, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 37

Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the calorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF₁₆₅ or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 38

Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 39

Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W., et al., J. Immunological Methods 1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 40

Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation:

2KNO₂+2KI+2H₂SO₄6 2NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding graded concentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing KI and H₂SO₄. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×10⁶ endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 41

Effect of Polypepides of the Invention on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from Cell Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications' Chord Formation Medium containing control buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quantitated through use of the Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-esteradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 42

Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old qual embryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 43

Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. The Matrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with the mixture of Matrigel and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the Matrigel plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). Matrigel alone is used to determine basal levels of angiogenesis.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 44

Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral arteries as described previously (Takeshita et al, Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.

The studies described in this example tested activity of polynucleotides and polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the agonists, and/or antagonists of the invention.

Example 45

Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean +/− SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 46

Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization.

The study in this model is divided into three parts as follows:

a) Ischemic skin

b) Ischemic skin wounds

c) Normal wounds

The experimental protocol includes:

a) Raising a 3×4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal).

b) An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap).

c) Topical treatment with a polypeptide of the invention of the excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100 mg.

d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding for histological, immunohistochemical, and in situ studies.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 47

Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:

a) One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoral artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 48

Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:

a) The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks.

c) Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 49

Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer.

b) Inserting a spatula below the lip of the incision facing the outer corner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide of the invention, within the pocket.

e) Treatment with a polypeptide of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 50

Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

A. Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer can serve as a positive tissue control and human brain tissue can be used as a negative tissue control. Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 51

Lymphadema Animal Model

or The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 34 weeks.

Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ˜350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then and ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.

Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (A J Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of ˜0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.

To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people then those 2 readings are averaged. Readings are taken from both control and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software (Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezegel, dipped into cold methylbutane, placed into labeled sample bags at −80EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 52

Suppression of TNF Alpha-Induced Adhesion Molecule Expression by a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

Tumor necrosis factor alpha (TNF-α), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.

The potential of a polypeptide of the invention to mediate a suppression of TNF-α induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-α treated ECs when co-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO₂. HUVECs are seeded in 96-well plates at concentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/nil streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× with PBS (+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Anti-ICAM-l-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS (+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS (+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰)>10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 53

Assay for the Stimulation of Bone Marrow CD34+ Cell Proliferation

This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of isolated polypeptides expressed in mammalian cells to stimulate proliferation of CD34+ cells.

It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of polypeptides on hematopoietic activity of a wide range of progenitor cells, the assay contains a given polypeptide in the presence or absence of other hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a “survival” factor. However, combined with any factor exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested polypeptide has a stimulatory effect on a hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect of a given polypeptide, or agonists or antagonists thereof, might not be detected. Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+IL+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.

Briefly, CD34+ cells are isolated using methods known in the art. The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md. Cat#160-204-101). After several gentle centrifugation steps at 200×g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5×10⁵ cells/ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with a given polypeptide in this assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μl SID (supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 μl. The plates are then placed in a 37° C./5% CO₂ incubator for five days.

Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H] Thymidine is added in a 10 μl volume to each well to determine the proliferation rate. The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OmniFilter assemblies consisting of one OmniFilter plate and one OmniFilter Tray. 60 μl Microscint is added to each well and the plate sealed with TopSeal-A press-on sealing film A bar code 15 sticker is affixed to the first plate for counting. The sealed plates is then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.

The studies described in this example test the activity of a given polypeptide to stimulate bone marrow CD34+ cell proliferation. One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof. As a nonlimiting example, potential antagonists tested in this assay would be expected to inhibit cell proliferation in the presence of cytokines and/or to increase the inhibition of cell proliferation in the presence of cytokines and a given polypeptide. In contrast, potential agonists tested in this assay would be expected to enhance cell proliferation and/or to decrease the inhibition of cell proliferation in the presence of cytokines and a given polypeptide.

The ability of a gene to stimulate the proliferation of bone marrow CD34+ cells indicates that polynucleotides and polypeptides corresponding to the gene are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein.

Example 54

Assay for Extracellular Matrix Enhanced Cell Response (EMECR)

The objective of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to identify gene products (e.g., isolated polypeptides) that act on the hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.

Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fn is mediated by the α₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM environment and responsible for stimulating stem cell self-renewal has not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications

Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fn fragment at a coating concentration of 0.2 μg/cm². Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 (5 ng/ml)+SCF (50 ng,/ml) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Gene products are tested with appropriate negative controls in the presence and absence of SCF (5.0 ng/ml), where test factor supernates represent 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment (5% CO₂, 7% O₂, and 88% N₂) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thymidine incorporation into cellular DNA. Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

If a particular gene product is found to be a stimulator of hematopoietic progenitors, polynucleotides and polypeptides corresponding to the gene may be useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein. The gene product may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

Additionally, the polynucleotides and/or polypeptides of the gene of interest and/or agonists and/or antagonists thereof, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.

Moreover, polynucleotides and polypeptides corresponding to the gene of interest may also be useful for the treatment and diagnosis of hematopoietic related disorders such as, for example, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

Example 55

Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation

The polypeptide of interest is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the polypeptide of interest on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity.

Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2%FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5%FBS. After incubation@37° C. for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μl/ml Amphotericin B, 0.4% FBS. Incubate at 37C until day 2.

On day 2, serial dilutions and templates of the polypeptide of interest are designed which should always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Then add ⅓ vol media containing controls or supernatants and incubate at 37 C/5% CO₂ until day 5.

Transfer 60 μl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4C until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10 μl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CytoFluor. This yields the growth stimulation/inhibition data.

On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 ul/well of Anti-Human L6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.

On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.

Wash plates with wash buffer and blot on paper towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover the plate and incubate 1 h at RT. Wash plates with wash buffer. Blot on paper towels.

Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.

A positive result in this assay suggests AoSMC cell proliferation and that the gene product of interest may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of polypeptides, polynucleotides, agonists and/or antagonists of the gene/gene product of interest. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, polypeptides of the gene product and polynucleotides of the gene may be used in wound healing and dermal regeneration, as well as the promotion of vasculargenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, antagonists of polypeptides of the gene product and polynucleotides of the gene may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, antagonists of polypeptides of the gene product and polynucleotides of the gene may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

Example 56

Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are then incubated at 37° C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min. Fixative is removed from the wells and wells are washed 1× with PBS (+Ca,Mg)+0.5% BSA and drained. 10 pi of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed three times with PBS (+Ca,Mg)+0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution, refered to herein as the working dilution) are added to each well and incubated at 37° C. for 30 min. Wells are washed three times with PBS (+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 μl of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰) >10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

Example 57

Alamar Blue Endothelial Cells Proliferation Assay

This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of the protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.

Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37-C overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the protein of interest or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C. incubator for three days. After three days 10 ml of stock alamar blue (Biosource Cat#DAL1100) is added to each well and the plate(s) is/are placed back in the 37° C. incubator for four hours. The plate(s) are then read at 530 nm excitation and 590 nm emission using the CytoFluor fluorescence reader. Direct output is recorded in relative fluorescence units.

Alamar blue is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form. i.e. stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity. The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.

Example 58

Detection of Inhibition of a Mixed Lymphocyte Reaction

This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides). Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by these polypeptides since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.

Polypeptides of interest found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of test materials (50 μl) is added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.

Samples of the protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

                   #             SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 170 <210> SEQ ID NO 1 <211> LENGTH: 733 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc cc #agcacctg     60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac ac #cctcatga    120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa ga #ccctgagg    180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aa #gccgcggg    240 aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg ca #ccaggact    300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca ac #ccccatcg    360 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac ac #cctgcccc    420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aa #aggcttct    480 atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aa #ctacaaga    540 ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ct #caccgtgg    600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat ga #ggctctgc    660 acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cg #acggccgc    720 gactctagag gat               #                   #                   #     733 <210> SEQ ID NO 2 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: Site <222> LOCATION: (3) <223> OTHER INFORMATION: Xaa equals any of the  #twenty naturally ocurring       L-amino acids <400> SEQUENCE: 2 Trp Ser Xaa Trp Ser   1               5 <210> SEQ ID NO 3 <211> LENGTH: 86 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 gcgcctcgag atttccccga aatctagatt tccccgaaat gatttccccg aa #atgatttc     60 cccgaaatat ctgccatctc aattag           #                   #              86 <210> SEQ ID NO 4 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 gcggcaagct ttttgcaaag cctaggc           #                   #             27 <210> SEQ ID NO 5 <211> LENGTH: 271 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5 ctcgagattt ccccgaaatc tagatttccc cgaaatgatt tccccgaaat ga #tttccccg     60 aaatatctgc catctcaatt agtcagcaac catagtcccg cccctaactc cg #cccatccc    120 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa tt #ttttttat    180 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt ga #ggaggctt    240 ttttggaggc ctaggctttt gcaaaaagct t         #                   #         271 <210> SEQ ID NO 6 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 gcgctcgagg gatgacagcg atagaacccc gg        #                   #          32 <210> SEQ ID NO 7 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 gcgaagcttc gcgactcccc ggatccgcct c         #                   #          31 <210> SEQ ID NO 8 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 ggggactttc cc               #                   #                   #       12 <210> SEQ ID NO 9 <211> LENGTH: 73 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact tt #ccatcctg     60 ccatctcaat tag               #                   #                   #      73 <210> SEQ ID NO 10 <211> LENGTH: 256 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 10 ctcgagggga ctttcccggg gactttccgg ggactttccg ggactttcca tc #tgccatct     60 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc ta #actccgcc    120 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg ca #gaggccga    180 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg ga #ggcctagg    240 cttttgcaaa aagctt              #                   #                   #   256 <210> SEQ ID NO 11 <211> LENGTH: 3289 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 11 cccgggtcga cccacgcgtc cggcgaggac cgcgtccggc gcagtcttca at #gagcagcg     60 cggaaactgc accccagacc cgagcctgct gcgcgccccc tcccagagct ca #cctggtgc    120 caggtaacag gcctggcctc gccctgtgga tgatgatggc cttgcccccg tg #agctacaa    180 cctggccttc agcacccgcc cacctccaac cagcaggatg cggctgtgga ag #gcggtggt    240 ggtgactttg gccttcatga gtgtggacat ctgcgtgacc acggccatct at #gtcttcag    300 ccacctggac cgcagcctcc tggaggacat ccgccacttc aacatctttg ac #tcggtgct    360 ggatctctgg gcagcctgcc tgtaccgcag ctgcctgctg ctggggagcc ac #cattggtg    420 tggccaagaa cagtgcgctg gggccccggc ggctgcgggc ctcgtggctg gt #catcaccc    480 tcgtgtgcct cttcgtgggc atctatgcca tggtgaagct gctgctcttc tc #agaggtgc    540 gcaggcccat ccgggacccc tggttttggg ccctgttcgt gtggacgtac at #ttcactcg    600 gcgcatcctt cctgctctgg tggctgctgt ccaccgtgcg gccaggcacc ca #ggccctgg    660 agccaggggc ggccaccgag gctgaaggct tccctgggag cggccggcca cc #gcccgaac    720 aagcgtctgg ggccacgctg cagaagctgc tctcctacac caagcccgac gt #ggccttcc    780 tcgtggccgc ctccttcttc ctcatcgtgg cagctctggg agagaccttc ct #gccctact    840 acacgggccg cgccattgat ggcatcgtca tccagaaaag catggatcag tt #cagcacgg    900 ctgtcgtcat cgtgtgcctg ctggccattg gcagctcatt tgccgcaggt at #tcggggcg    960 gcatttttac cctcatattt gccagactga acattcgcct tcgaaactgt ct #cttccgct   1020 cactggtgtc ccaggagaca agcttctttg atgagaaccg cacaggggac ct #catctccc   1080 gcctgacctc ggacaccacc atggtcagcg acctggtctc cagaacatca at #gtcttcct   1140 gcggaacaca gtcaaggtca cgggcgtggt ggtcttcatg ttcagcctct ca #tggcagct   1200 ctccttggtc accttcatgg gcttccccat catcatgatg gtgtccaaca tc #tacggcaa   1260 gtactacaag aggctctcca aagaggtcca gaatgccctg gccagagcga gc #aacacggc   1320 ggaggagacc atcagtgcca tgaagactgt ccggagcttc gccaatgagg ag #gaggaggc   1380 agaggtgtac ctgcggaagc tgcagcaggt gtacaagctg aacaggaagg ag #gcagctgc   1440 ctacatgtac tacgtctggg gcagcgggct cacactgctg gtggtccagg tc #agcatcct   1500 ctactacggg ggccaccttg tcatctcagg ccagatgacc agcggcaacc tc #atcgcctt   1560 catcatctac gagtttgtcc tgggagattg tatggagaat gtctccttca gc #ctgtcccc   1620 cggcaaggtg acggccctgg tggggccctc gggcagtggg aagagctcct gt #gtcaacat   1680 cctggagaac ttctaccccc tggagggggg ccgggtgctg ctggacggca ag #cccatcag   1740 cgcctacgac cacaagtact tgcaccgtgt gatctccctg gtgagccagg ag #cccgtgct   1800 gttcgcccgc tccatcacgg ataacatctc ctacggcctg cccactgtgc ct #ttcgagat   1860 ggtggtggag gccgcacaga aggccaatgc ccacggcttc atcatggaac tc #caggacgg   1920 ctacagcaca gagacagggg agaagggcgc ccagctgtca ggtggccaga ag #cagcgggt   1980 ggcatggccc gggctctggt gcggaacccc ccagtcctca tcctggatga ag #ccaccagc   2040 gctttggatg ccgagagcga gtatctgatc cagcaggcca tccatggcaa cc #tgcagaag   2100 cacacggtac tcatcatcgc gcaccggctg agcaccgtgg agcacgcgca cc #tcattgtg   2160 gtgctggaca agggccgcgt agtgcagcag ggcacccacc agcagctgct gg #cccagggc   2220 ggcctctacg ccaagctggt gcagcggcag atgctggggc ttcagcccgc cg #cagacttc   2280 acagctggcc acaacgagcc tgtagccaac ggcagtcaca aggcctgatg gg #gggcccct   2340 gcttctcccg gtggggcaga ggacccggtg cctgcctggc agatgtgccc ac #ggaggccc   2400 ccagctgccc tccgagccca ggcctgcagc actgaaagac gacctgccat gt #cccatgga   2460 tcaccgcttc ctgcatcttg cccctggtcc ctgccccatt cccagggcac tc #cttacccc   2520 tgctgccctg agccaacgcc ttcacggacc tccctagcct cctaagcaaa gg #tagagctg   2580 cctttttaaa cctaggtctt accagggttt ttactgtttg gtttgaggca cc #ccagtcaa   2640 ctcctagatt tcaaaaacct ttttctaatt gggagtaatg gcgggcactt tc #accaagat   2700 gttctagaaa cttctgagcc aggagtgaat ggcccttcct tagtagcctg gg #ggatgtcc   2760 agagactagg cctctcccct ttacccctcc agagaagggg cttccctgtc cc #ggagggag   2820 acacggggaa cgggattttc cgtctctccc tcttgccagc tctgtgagtc tg #gccagggc   2880 gggtagggag cgtggagggc atctgtctgc catcgcccgc tgccaatcta ag #ccagtctc   2940 actgtgaacc acacgaaacc tcaactgggg gagtgagggg ctggccaggt ct #ggaggggc   3000 ctcaggggtg cccccagccc ggcacccagc gctttcgccc ctcgtccacc ca #cccctggc   3060 tggcagcctc cctccccaca cccgcccctg tgctctgctg tctggaggcc ac #gtggatgt   3120 tcatgagatg cattctcttc tgtctttggt ggatgggatg gtggcaaagc cc #aggatctg   3180 gctttgccag aggttgcaac atgttgagag aacccggtca ataaagtgta ct #acctctta   3240 cccctaaaaa aaaaaaaaaa aaaaaaaaaa aaagggcggc cgctctaga   #             3289 <210> SEQ ID NO 12 <211> LENGTH: 2342 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 12 ggcacgagct cagatctctc ctggtacccc ttccccacgc ccttagataa tc #catctcaa     60 ttcctcatgc taattgagga gctatggctg caaggcacct tccaggattt ca #cacctaca    120 caaatctcct ttttctcctt ttgccttctc tgcttatggg atattctgag tc #cccacccc    180 caatcactga cagctgggcc cccttcatca gcctcacaca ccacgtatta ag #tcagtcac    240 aatckcccct ctcmtctaac tgctggattt gtctttctac acacacccaa tg #attcacgg    300 ctcttccggc tgamctactt acatggacac agtccaacgt gagcttacac at #ttcttact    360 tggctatacc attcttggct gattcattcc tgaaamcggt tcataacctg ga #aactcagc    420 taaacacctt tccttcaaac tcagctctct cagcatggtt tcaggtaggg ct #gttgccct    480 ccttcaccta atagcttctg gactaacatc catacaaacc aacacagcat ca #tctaaacc    540 accaatatgg gggtaccatt tctactcaaa cttccttcat atccccaccc cc #cttatgtc    600 tcagccgaac ctaccctaat ccagcccacg ccacaatggt gggacaggtt cc #ccagtccc    660 tatgtggtct tatttttacc cttgcactcc ctgtagacca tcaattctac ac #cctaatta    720 caaaatcata tccacctctg cctggcagaa ggtgttatgc ttttctggct cg #cctaccat    780 ccacacatcc ctacacctca ccaccggatc ctcttttctt tccttccatc ca #attcctgg    840 cttccccgct gccaactctg ctctctatgt ctccagttta aaggtgcccc ct #ggaaaaaa    900 tgtaacaatt ccctcacctg tgactggtac ctgacagcca ccacaccggg gc #agcaatgg    960 ctaacggttg acaaagacaa tttctttctc tctccaaaac caaacagcct tc #atcaactc   1020 cctagccaag actccctatc aggcccttac aggtgccgct ctggctggca gt #tacccaat   1080 ttgggaaaac gaaaataccc tatcatggta cctaccttca cctacaactt tg #ctgtcacc   1140 cccagttctc ttttgtgtga tacaactgat atttkgccta ccagccaact gg #tcaggaac   1200 ttgcaccctg gtctttcagg ctccaaccat caacatccta ccccctaacc aa #actattct   1260 aatttctgta gaagcctcta tctcctcttc acccataaga aataaatggg ct #ctacatct   1320 catcaccctg ctaacaggat taggcatcac tgctgcactt ggcactggaa ta #gcaggcat   1380 aaccacctca atcacctcat accaaacact attcacaacc ctttctaaca cc #gtagaaga   1440 tatgcacact tccattacca gtctccaacg acaattagac ttcctcgtgg ga #gtcatcct   1500 tcaaaactgg agagtcctgg acctcctaac cactgagaaa gggggtacct gc #atatacct   1560 ccaggaagaa tgctgtttct gtgttaatga atctggcatt gttcatatcg ca #gttcgtag   1620 gcttcatgac agggctgcag agctttgaca tcaagtcgct gactcctggt gg #caaggatc   1680 atcccttcta agatggatac cctgggttgc ccccttccta ggacccctga tc #ttcctctt   1740 cctgttacta atgattgggc catgcatatt taaccttgta tcccgcttca tt #tcccaaag   1800 gctgaattgt tttatccagg caagcatgca aaaacacatt gataatatat tt #cacctttg   1860 ccacgtctaa taccagagcc tacgaggaaa ccattcggaa gctccagaac cc #aggcccta   1920 atcacaacgc ccctatccag caggaagcag ccagatgatc aacgacgccc tt #tttccttt   1980 ttatactaaa gtaagaaata agaatgttag cccaaactgc actattttgc ag #acccctac   2040 cattttacaa actggtcaga gtggaaaatt ccaccagggc ctgagctgtg ag #aaacatcc   2100 tgtcaggcag gtcccaggcc taacccctgg ctgcactaaa ttccttcatt at #cagcagcc   2160 aaacacaccg cccccacccc attttcacaa caatcccaga cctctcctgc cc #gggactgt   2220 aactggtcca gcctgtaagc gggaaggggg ctctggcact agctggtacc cc #ctctccgc   2280 aggtctttct cccaataaat ctgtgttgcc attgaaaaaa aaaaaaaaaa aa #aaaaactc   2340 ga                   #                   #                   #            2342 <210> SEQ ID NO 13 <211> LENGTH: 1666 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 ggtggagttc gcacctccag ctcgggccga tgtggaagct ttggagagct ga #agagggcg     60 cggcggcgct cggcggcgcg ctcttcctgc tgctcttcgc gctaggggtc cg #ccagctgc    120 tgaagcagag gcggccgatg ggcttccccc cggggccgcc ggggctgcca tt #tatcggca    180 acatctattc cctggcagcc tcatccgagc ttccccatgt ctacatgaga aa #gcagagcc    240 aggtgtacgg agagatcttc agtttagatc ttggaggcat atcaactgtg gt #tctaaatg    300 gctatgatgt agtaaaggaa tgccttgttc atcaaagcga aatttttgca ga #cagaccat    360 gccttccttt attcatgaag atgacaaaaa tgggaggctt actcaattcc ag #atatggcc    420 gaggatgggt tgatcacaga cgattagctg taaacagttt tcgatatttt gg #atatggcc    480 aaaagtcttt tgaatctaaa atcttggaag aaaccaaatt tttcaatgat gc #tattgaaa    540 catacaaagg tagacctttt gactttaaac agttaataac gaatgctgtt tc #aaacataa    600 ccaatctgat catttttgga gaacgattca cttatgaaga caccgatttt ca #gcacatga    660 ttgagttatt tagtgaaaat gtggaactag ctgccagtgc ctcagtcttc tt #gtataatg    720 cctttccatg gattggcatc ctgccttttg gaaaacatca acagctgttt ag #aaatgcag    780 ctgtagtcta tgattttctc tccagactca ttgaaaaagc ttcagtcaac ag #aaagcctc    840 agctacctca gcattttgtt gatgcttatt tagatgagat ggatcaaggt aa #aaatgacc    900 catcatctac tttctccaaa gaaaacctaa ttttctcagt gggtgaactc at #cattgctg    960 gaactgaaac tacaaccaat gtgctacggt gggcgattct tttcatggcc ct #ttatccta   1020 atattcaagg acaagttcag aaagagattg atttaattat gggccctaat gg #gaagcctt   1080 cttgggacga caaatgcaaa atgccttata ctgaggcagt tttgcatgaa gt #tttaagat   1140 tctgtaatat agttccatta gggattttcc atgcaacctc tgaagatgca gt #tgtacgtg   1200 gttattccat tcctaaaggc acaacagtaa ttacaaatct ttattctgta ca #ctttgatg   1260 aaaagtactg gagagaccca gaagtgttcc atcctgagcg atttctggac ag #cagtggat   1320 attttgccaa gaaggaagct ttggttcctt tttccctagg aagaagacat tg #tcttggag   1380 aacacttggc tcggatggaa atgttcttgt tttttacagc attgcttcag ag #gtttcatt   1440 tgcattttcc acatgaacta gttccagatc tgaagcccag gttaggcatg ac #attgcagc   1500 cccaacccta cctcatctgt gctgaaagac gctgaaactg cctgggatgt tt #tcgggaac   1560 aagaatgtat atttgcctta tccctgaact tggtttaatc aaatcaatgt gt #gtattaga   1620 ataaaagtca cagcatcaaa aagmcaaaaa aaaaaaaaaa aaaaaa    #               1666 <210> SEQ ID NO 14 <211> LENGTH: 2027 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (294) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1976) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1981) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1985) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (2021) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 14 ggcacgagtt gggagcagct ctgcgtgcgg ggcctcagag aatgaggccg gc #gttcgccc     60 tgtgcctcct ctggcaggcg ctctggcccg ggccgggcgg cggcgaacac cc #cactgccg    120 accgtgctgg ctgctcggcc tcgggggcct gctacagcct gcaccacgct ac #catgaagc    180 ggcaggcggc cgaggaggcc tgcatcctgc gaggtggggc gctcagcacc gt #gcgtgcgg    240 gcgccgagct gcgcgctgtg ctcgcgctcc tgcgggcagg cccagggccc gg #anggggct    300 ccaaagacct gctgttctgg gtcgcactgg agcgcaggcg ttcccactgc am #cctggaga    360 acgagccttt gcggggtttc tcctggctgt cctccgaccc cggcggtctc ga #aagcgaca    420 cgctgcagtg ggtggaggag ccccaacgct cctgcaccgc gcggagatgg gt #acttccag    480 gccaccggtg gggtcgagcc cgcagctgga aggagatgcg atgccacctg yg #cgccaacg    540 ctacctgtgc aagtaccagt ttgaggtctt gtgtcctgcg ccgcgccccg gg #gccgcctc    600 taacttgagc tatcgcgcgc ccttccagct gcacagcgcc gctctggact tc #agtccacc    660 tgggaccgag gtgagtgcgc tctgccgggg acagctcccg atctcagtta ct #tgcatcgc    720 ggacgaaatc ggcgctcgyt gggacaaact ytcgggcgat gtgttgtgtc cc #tgccccgg    780 gaggtacctc cgtgctggca aatgcgcaga gctccctaac tgcctagacg ac #ttgggagg    840 ctttgcctgc gaatgtgcta cgggcttcga gctggggaag gacggccgct ct #tgtgtgac    900 cagtggggaa ggacagccga cccttggggg gaccggggtg cccaccaggc gc #ccgccggc    960 cactgcaacc agccccgtgc cgcagagaac atggccaatc agggtcgacg ag #aagctggg   1020 agagacacca cttgtccctg aacaagacaa ttcagtaaca tctattcctg ag #attcctcg   1080 atggggatca cagagcacga tgtctaccct tcaaatgtcc cttcaagccg ag #tcaaaggc   1140 cactatcacc ccatcaggga gcgtgatttc caagtttaat tctacgactt cc #tctgccac   1200 tcctcaggct ttcgactcct cctctgccgt ggtcttcata tttgtgagca ca #gcagtagt   1260 agtgttggtg atcttgacca tgacagtact ggggcttgtc aagctctgct tt #cacgaaag   1320 cccctcttcc cagccaagga aggagtctat gggcccgccg ggctggagag tg #atcctgaa   1380 gcccgctgct ttgggctcca gttctgcaca ttgcacaaac aatggggtga aa #gtcgggga   1440 ctgtgatctg cgggacagag cagagggtgc cttgctggcg gagtcccctc tt #ggctctag   1500 tgatgcatag ggaaacaggg gacatgggca ctcctgtgaa cagtttttca ct #tttgatga   1560 aacggggaac caagaggaac ttacttgtgt aactgacaat ttctgcagaa at #cccccttc   1620 ctctaaattc cctttactcc actgaggagc taaatcagaa ctgcacactc ct #tccctgat   1680 gatagaggaa gtggaagtgc ctttaggatg gtgatactgg gggaccgggt ag #tgctgggg   1740 agagatattt tcttatgttt attcggagaa tttggagaag tgattgaact tt #tcaagaca   1800 ttggaaacaa atagaacaca atataattta cattaaaaaa taatttctac ca #aaatggaa   1860 aggaaatgtt ctatgttgtt caggctagga gtatattggt tcgaaatccc ag #ggaaaaaa   1920 ataaaaataa aaaattaaag gattgttgat aaaaaaaaaa aaaaagggcg gc #cgcnctag   1980 ngggnccaag ctttacgtac gcgggcatgc gacgtcaagc ncttcca    #              2027 <210> SEQ ID NO 15 <211> LENGTH: 2334 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (2278) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (2290) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 15 gggagtgtgg ctgcagaacc caggtggcag ggctttcctc aggcccctta ct #cctgacct     60 ggacgaggcc ggggcttcct caaggaggct ctctgactgc cacccctgcc tg #cctgcccg    120 gccctgcaca acatgcagcc ctccggcctc gagggtcccg gcacgtttgg tc #ggtggcct    180 ctgctgagtc tgctgctcct gctgctgctg ctccagcctg taacctgtgc ct #acaccacg    240 ccaggccccc ccagagccct caccacgctg ggcgccccca gagcccacac ca #tgccgggc    300 acctacgctc cctcgaccac actcagtagt cccagcaccc agggcctgca ag #agcaggca    360 cgggccctga tgcgggactt cccgctcgtg gacggccaca acgacctgcc cc #tggtccta    420 aggcaggttt accagaaagg gctacaggat gttaacctgc gcaatttcag ct #acggccag    480 accagcctgg acaggcttag agatggcctc gtgggcgccc agttctggtc ag #cctatgtg    540 ccatgccaga cccaggaccg ggatgccctg cgcctcaccc tggagcagat tg #acctcata    600 cgccgcatgt gtgcctccta ttctgagctg gagcttgtga cctcggctaa ag #ctctgaac    660 gacactcaga aattggcctg cctcatcggt gtagagggtg gccactcgct gg #acaatagc    720 ctctccatct tacgtacctt ctacatgctg ggagtgcgct acctgacgct ca #cccacacc    780 tgcaacacac cctgggcaga gagctccgct aagggcgtcc actccttcta ca #acaacatc    840 agcgggctga ctgactttgg tgagaaggtg gtggcagaaa tgaaccgcct gg #gcatgatg    900 gtagacttat cccatgtctc agatgctgtg gcacggcggg ccctggaagt gt #cacaggca    960 cctgtgatct tctcccactc ggctgcccgg ggtgtgtgca acagtgctcg ga #atgttcct   1020 gatgacatcc tgcagcttct gaagaagaac ggtggcgtcg tgatggtgtc tt #tgtccatg   1080 ggagtaatac agtgcaaccc atcagccaat gtgtccactg tggcagatca ct #tcgaccac   1140 atcaaggctg tcattggatc caagttcatc gggattggtg gagattatga tg #gggccggc   1200 aaattccctc aggggctgga agacgtgtcc acatacccag tcctgataga gg #agttgctg   1260 agtcgtggct ggagtgagga agagcttcag ggtgtccttc gtggaaacct gc #tgcgggtc   1320 ttcagacaag tggaaaaggt acaggaagaa aacaaatggc aaagcccctt gg #aggacaag   1380 ttcccggatg agcagctgag cagttcctgc cactccgacc tctcacgtct gc #gtcagaga   1440 cagagtctga cttcaggcca ggaactcact gagattccca tacactggac ag #ccaagtta   1500 ccagccaagt ggtcagtctc agagtcctcc ccccacatgg ccccagtcct tg #cagttgtg   1560 gccaccttcc cagtccttat tctgtggctc tgatgaccca gttagtcctg cc #agatgtca   1620 ctgtagcaag ccacagacac cccacaaagt tcccctgttt gcaggcacaa at #atttcctg   1680 aaataaatgt tttggacata gaaaaaaaaa aaaaaaaaag ggcggccgct ct #agaggatc   1740 cctcgagggg cccaagctta cgcgtgcatg cgacgtcata gctctctccc ta #tagtgagt   1800 cgtattataa gctaggcact ggccgtcgtt ttacaacgtc gtgactggga ga #tctgctag   1860 cttgggatct ttgtgaagga accttacttc tgtggtgtga cataattgga ca #aactacct   1920 acagagattt aaagctctaa ggtaaatata aaatttttaa gtgtataatg tg #ttaaacta   1980 gctgcatatg cttgctgctt gagagttttg cttactgagt atgatttatg aa #aatattat   2040 acacaggagc tagtgattct aattgtttgt gtattttaga ttcacagtcc ca #aggctcat   2100 ttcaggcccc tcagtcctca cagtctgttc atgatcataa tcagccatac ca #catttgta   2160 gaggttttac ttgctttaaa aaacctycca cacctccccc tgaacctgaa ac #ataaaatg   2220 aatgcaattg gtggtggtaa cttggttaat ggagcttata atggtaccaa ta #aagcantg   2280 catcacaaan ttcccaaata aagcattttt tcctggaatt taaatggggg tt #gg         2334 <210> SEQ ID NO 16 <211> LENGTH: 2608 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 ccacgcgtcc gcacagggct caggctgggc tcagaggcca ctgagatgcc ag #ctccttga     60 gagcagtggg ggtgtcccag gccagagcag cctcttcctc tcctggtccc ag #aaaaaccc    120 ttgcagtaaa tggtggcctc tgggtggtta cttctagcgc aggcttcctt cc #ttcctcta    180 gctcctcccg gtgccctggg tgctgggtgc tggatggatg ggcgtcctct ag #ctcctccc    240 ggtgccctgg gtgctgggtg ctggatgggt gggcgtcctc tagctcctcc cg #gtgccctg    300 ggtgctgggt gctggatggg tgggcgccac ggtgcaccct tgttgggctg cc #tgtgcccg    360 agtggcctct gcagctctta tgtctgcctc taatgggatg tgcgccctga ct #gcctcgtt    420 cttaagggca tagtgggtcg gctaagatct gatcgccagg actgcgttct gg #gcaggtgc    480 tgggaaggcg gaaccaagcc gtccgtgccg ctagggagcc gagactgccg ga #aagaagag    540 cggcaggagg gggcgtggtt gtgcagcccc acccccggga ggggctttag gc #actgggaa    600 ggaaagtcct gttggaggaa ttcggtggct gttcacaccc gcctcgctgt ct #tggagtct    660 tgatctgtcg tcggcgggtc gctggcacag gactaaacat ggctgaggct gg #gctccagc    720 cagatctggc tggggacagc accgcgtggg cccaggatcc acccagagca gg #caggcctt    780 gctggggccc cagtcaagtc cacttccagt gaggagagcc agccgggagg tc #agtgccag    840 agctctggtg gagcccagac cctgccttcc ctgagggccg cccctgtcgc cg #ccctgggg    900 tccctgtcct cctatcctga ctcctgcccc agggccacca cccctgaact gt #gccctggg    960 gcccccaccc tccacctggc cgactccatc tctgggcctg tcagtccacc tg #ggtcctct   1020 ctgggccctg atgcctggac cctctgtgcc aagcaccacc aagcaaaggg ga #tgaccttg   1080 ggcaccccca aggtgctgag actacagcca gtgagcccct gctgggggcc aa #agtcatgg   1140 agggtgcctg ggcccttcca acctggaagg aggaggggag agagcaggca gc #agggcagg   1200 gggaagagga ggagtgcccg atctgcacag agccctacgg gcccagagag cg #ccgcctgg   1260 ccctgctgaa ctgtagccac ggcctgtgtg tgggctgcct gcacaggctg ct #gggctcgg   1320 cctccagtgc cgacctgggc cgggtgcgct gcccgctgtg ccgcagaaga cg #cccgtgct   1380 ggagtgggag atctgccggc tacaggagga gctgctacag gccgacgggc cc #tcacgcca   1440 gccccgccga gaggcccctg catcctatca ccgcaaccct gggccctggg gc #tccctgga   1500 gcaccgctac cagctgcgct tcctggcagg gcccgtgggc ggccggggct gc #ctgccctt   1560 cctgccctgt ccaccctgcc tgggtgcccg gctctggacc ctgcgggagc gg #ggaccctg   1620 tgcccgccgc ctggcgctgc tgagcctgct ggcccttgag cttctggggc tg #ctgctggt   1680 cttcacgccg ctcctgctgc tgggactgct cttcgtgctc ctggaccgct ct #ggccgctg   1740 agcagagccc aggacagccc cgccgcaaca ggccaggggg cccagactgg cc #cacgtccc   1800 catgcctggg tgctgtgagg cctgatgacc aggctggaaa aacccaaggt tg #ggtccagg   1860 gcagtggcct tcaatcaaga cctcccattg ctgaacccac aaccagggct ac #ccagaggc   1920 ctgaccctgc agagtccatg gctgcactgc tgcccagaca ctagctgaac cc #aaggacac   1980 cagcgcccaa ggacagctcc tggaggaggc cagcccagca ggaaagtctg tg #agcaggac   2040 cccattcacc ctgcggcaga cgggcaccgt actggccacg ggctgacgcc gg #ccacactt   2100 cccctccgag ggccagctga gcacagcagg catgaaagca aacagagata ca #gcagtgag   2160 tcagttcctt ggagagggca gggactccgc ccaccctgtg ttcagataag gg #ccagtgtg   2220 tgtccctgaa ggtcaggcca gccgggggag gggtccatgc tgcgaaaatt ca #gcctgcaa   2280 aggctcctct ccccacttga tcaggcccag accaggtggg ggttggcgct gg #cctgtgtt   2340 gcaggggaca agggcccacc caggccttgg aacataagct ctgcccctgc ac #accctcat   2400 gtcaccacac ctgggatgga gacatcaggt ggcccagcga gagatggagg ac #tgatcctg   2460 gaacgtgaag cagctttcaa taaaccagct cctggggaaa aaaaaaaaaa aa #aaaaaaaa   2520 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa #aaaaaaaa   2580 aaaaaaaaaa aaaaaaaaaa aaaaaaaa          #                   #           2608 <210> SEQ ID NO 17 <211> LENGTH: 1291 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1279) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1286) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1290) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 17 aaacctcttc tataggtaaa gctggwacgc ctgcaggtac cggtccggaa tt #cccgggtc     60 gcccacgcgt ccggaaagag gaaacataga ggtgccaaag gaacaaagac at #aatgatgt    120 catccaagcc aacaagccat gctgaagtaa atgaaaccat acccaaccct ta #cccaccaa    180 gcagctttat ggctcctgga tttcaacagc ctctgggttc aatcaactta ga #aaaccaag    240 ctcagggtgc tcagcgtgct cagccctacg gcatcacatc tccgggaatc tt #tgctagca    300 gtcaaccggg tcaaggaaat atacaaatga taaatccaag tgtgggaaca gc #agtaatga    360 actttaaaga agaagcaaag gcactagggg tgatccagat catggttgga tt #gatgcaca    420 ttggttttgg aattgttttg tgtttaatat ccttctcttt tagagaagta tt #aggttttg    480 cctctactgc tgttattggt ggatacccat tctggggtgg cctttctttt at #tatctctg    540 gctctctctc tgtgtcagca tccaaggagc tttcccgttg tctggtgaaa gg #cagcctgg    600 gaatgaacat tgktagttct atcttggcct tcattggagt gattctgctg ct #ggtggata    660 tgtgcatcaa tggggtagct ggccaagact actgggccgt gctttctgga aa #aggcattt    720 cagccacgct gatgatcttc tccctcttgg agttcttcgt agcttgtgcc ac #agcccatt    780 ttgccaacca agcaaacacc acaaccaata tgtctgtcct ggttattcca aa #tatgtatg    840 aaagcaaccc tgtgacacca gcgtcttctt cagctcctcc cagatgcaac aa #ctactcag    900 ctaatgcccc taaaagaaaa aggggtatca gtctaatctc atggagaaaa ac #tacttgca    960 aaaacttctt aagaagatgt cttttattgt ctacaatgat ttctagtctt ta #aaaactgt   1020 gtttgagatt tgtttttagg ttggtcgcta atgatggctg tatctccctt ca #ctgtctct   1080 tcctacatta ccactactac atgctggcaa aggtgaagga tcagaggact ga #aaaatgat   1140 tctgcaactc tcttaaagtt agaaatgttt ctgttcatat tactttttcc tt #aataaaat   1200 gtcattagaa acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagggcg gc #cgctctag   1260 aggatccaag cttacgtang cgtgcntgcn a         #                   #        1291 <210> SEQ ID NO 18 <211> LENGTH: 3129 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 cggcacgagg ggagaccgaa tggcggggtg aggggagcta ctgcgtccgg ct #ttgcaggc     60 cgttcctttc agggttgccg cagcaatggc acttcatcct ggcagcagcc at #ttgctggt    120 tgcagtgcca gtttcttggt ttcttttttg cattcctgga atcagtttca tc #actctctc    180 ttggagttac caagagtcgc cagtcagttt cctctccgtg gaaggctgag tc #ccaattcc    240 atagggctct gtgccagact tttagtctct gataactccg aaatccttcc tt #ctgtttcc    300 taagctctac aggtattcta gtttttctcg caatttttca ttactaggct ac #atcagtgt    360 cctttttctt tctttctttt tctcactttt tatttgtacc tttagtcctc ca #acaattaa    420 caattcccca tgttaaattc tctctgttta aacaactaac gggctctctg ta #ttccaact    480 agatctcggc tgatgcaaat gccgtagaca atcaatcgat caatcaatca gc #aaatccaa    540 tttccctcta gccactaaag ggacttaata taattgaatg attataaaat tc #tgatccca    600 tttatctcaa gccattgaaa atatttttcg tgtgattata agatacgtaa tt #ttaaaaaa    660 tgattttcta aatcaggatt cttaccagat tatttcacag cttattcact ca #ttgattgc    720 tttcctccca cccttgttag cttttgtatg cctgtcctgc attttaaacc aa #ggtatgag    780 gaggataaag taaatagtaa atggtatgat agtggccaaa actttgaatc tt #tttcccta    840 ttcatcatct ttgtgcaagt tggtagggag gtccactaat tcagcctccc ca #ttccctgt    900 cccttcaaag taaatcagaa gcttcaggta aaaagcagag aagctaccca ag #ttcttttc    960 atttatttta tcatcatact aataattttg ctttctgaaa tgattactct ta #caagcagc   1020 aaatatattt tttaggaagt tagaattatt gatgtcacgt tttcatattc ag #tatcccaa   1080 gtaaatatca ggtaaattca ggaaaataaa tacaaccatt ttcttggtta at #tgttttta   1140 aatttaatat atgttcattt attcttttaa atatatattt cataaatggt ga #ctatgtat   1200 tccatgacat aaaaactaag tatttttagt gggaaattct ttgtggaatc ac #tgaaatga   1260 taactagtgt tagcaatact tctgtttcat gagacgagtc actttatgaa ga #ttgcaaat   1320 tattttttgt cccctctaga gttcatttca tgggaaattt aaattttatt gt #cttaactt   1380 taatgttaaa catttccttc acatatggta tggccagaca tgttttcttc tt #tgctgcta   1440 ttttacattt tagatgtagt ccattcagga tctttttttt ttttttttag ga #gtcagagc   1500 ttgaagtgat gcaacacttt cactctttat ctctgattta catgctaata aa #gacaactg   1560 atagacaata tgatatactt tggccctaga agatccttaa tgaggaaatt ct #gtggcctg   1620 ttttctcttc tgtgctattt tttgtcttct aaggcaggca tttttatcag tt #cagtttgt   1680 tgctgtgact agttcaaact tgttcagggc tttatgcctg aaataagtaa tc #aattagcc   1740 tatatatata aatatataac caaccattac aattatctaa gaatgtcagg ga #gttgtgaa   1800 gtggaaacaa attaaagagt tgcaaaatta aaagagctcg tcttccaaca ga #agtgtgct   1860 gacatatatt aaagaataaa tgaattaaag tcaccaaaga ggaacaggag tt #aaaaagaa   1920 gaccattgtt tagatggagt caggctggtg cactgccaag gaaagcaaac ag #gatttctg   1980 aaggcttggt atgaatttca gcttgcatca ttccgctgga gggggtgatt tc #cccaatat   2040 catgttagat ccatagcttg ttcttgacag agtggcagta cctttcctcc ac #tgcactct   2100 caccactagc atagatgtaa aacacagtaa gtactcagaa actacttgaa ga #gtgcagtt   2160 atcagtagag atgatcgaaa catttgtttt tctagggaat atttttgcct tt #cttcttcc   2220 agaatcctct ggttataatg tgctcactgc taggtcacca gtcataaaac at #tatgtaga   2280 ggttactggt cattatccta atatatttat caaaaaatct ggagtatatg aa #actgcctt   2340 tcattgtaac attagacaaa aacatttatt ctatcaaata cagacttaaa ac #tgccacca   2400 aattggaaga atatgatctt aaatttaaaa aaaccccata tacttaaaca ca #ataatcta   2460 tctttatcta ccttcctaaa cattaatgca tgagacttct cttaatatca tg #aatatgca   2520 ctatgatttt tatgttacat ctttttcttg tttccattta tgctagtgaa at #ttattagt   2580 atccttcatt gaaacactat tattttccat ggaggaaaat attttatttc ct #ttatgaaa   2640 agggtgcttt acttctgaaa tacagaattc attttgtttc ggattctgtt tg #tttgtttt   2700 cacatcaact tcattctaaa tgttcattca aaattatttt tcagacttga gt #tcaggtga   2760 aaatgttaat ggaattaaaa agtagttagt caaatgaaat ttcaatatat aa #gtcaaatt   2820 tgaagaaaac tgaattaata aaggattcta agtttataaa gaaatcaaaa tt #atgctttc   2880 aaaaatatta tgtaactgga gaaagtaatt ttattttgag atctctcatg at #tctttaat   2940 atatattttt tcttattagt atgcaacctt ttggtaacat atataggaga ca #gttaattt   3000 tggtaagact acataattca tacattcttc atcttgacat acatgatcaa at #actttata   3060 acgtccacta gccttgctct tgtccactgc aatggaaaaa aaaaaaaaaa aa #ctcgaggg   3120 ggggcccgg                 #                   #                   #       3129 <210> SEQ ID NO 19 <211> LENGTH: 3629 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 ccacgcgtcc gccagaggct gggaaggaaa aattattccc atcttaaaat at #cttaaaaa     60 tatgtacaga tttattgtaa atcaaaattt tcctcataac tacagcccct tc #ccacacat    120 aaaattcccc ctaaatcagg aacataatgt acagcctgtt tttaacttgt at #ttttccat    180 tcacactatg tcacaaaaaa attctcatgg tgatccatga tttcacaggt cc #tgtccatg    240 tgtttcctga gaagacagtc ctggaatgga attactaatt caaaaggcat tt #ttaaggct    300 gttacacata ctgccaaatc ggccttcagg aggatgacac aaattccacc ta #tgtcatct    360 gagcgtgcca ctatctgctg ctattgcccc atgtttgagg ttttgtcctt tc #tcctcatt    420 ctcatgggca actgtcaaca tcaccaccac tgtgatcctg aaaagaaagg ct #cagaagac    480 atagacagtc tgggattttt ttttttccct acctttcttt gcactgtagg ga #aggagtga    540 gatgtggctg caggattggg gaatagctaa gtcagaaatg agcagcagtg ca #actgtggc    600 aatcttggag gggggtctca gccatgaaag aaaacagggg gtgccttgcc ag #ctggaggc    660 tgcagaatct tagcttgggt gttttgcctt ctccgtggaa ctgacagatg ac #aggaatga    720 cgtggagtat tctcatcaag cgtctcagca tagtcagtct ctgagatggc aa #actttagg    780 caaagacacc tgggctgaaa ggtcccacat ttaaaacttg tcagtcagat aa #ctgcccac    840 caccaccctc tgtaagatac ctagatatgg gaaccccaca ctcttcccca at #aagactct    900 tgtatgactg aaagactttg tggttaaaaa catatcttaa tctaacctgt at #tttccctg    960 ctgcaatttc agccttttcc tatcttttca tggtgaatga acaccacaca gg #ctttcaga   1020 tcagacaact tctggctccg tcatttacta gctgtatcag ttaacaccag ct #gctgactt   1080 cacttctgca tatgcattcc aaaattagtg tcttaaaata acaactactt at #tacttttc   1140 acaagcctaa aagttggctg aatggttctg ctgatctgac ttggttaatc tt #ggctggac   1200 actgtctgat tcagcatagg ttgactgtcc tctgcctcat gtctcttatc at #ccataaat   1260 tgcccaggct tgcttgcctg aaggcagaag agttcccact agcaggagag gg #caagcact   1320 tttcaatgca caagcacctt tcaagcctct atttgcatca agtttgctaa tg #tcccgctg   1380 accaaagcaa atcatgtggc ttacttcgga gtcagtgtgg gtaggcatta cc #caaagaaa   1440 gtggctatag ggaagcatga agatctagag ccattagtac aatcaatcta cc #acattaac   1500 tttgtgacct tgggcaattc ccttagcctc tctgagcttt aactctataa tc #tataaaag   1560 tgcagaattt ttttgggatt ctttgctata atgtatgtaa aatgtctgct ac #acaataaa   1620 catgccatat ttattaattt tcttttctcc tccttttcat catttgaatt tt #ttcttaaa   1680 tgcatatata tatgtgcata tatatatgtc catctcgata tatatatatg ta #tatatata   1740 tatatatatg acttactctg tcatccaggt gagagtgcag tggcatgatc ac #agctcact   1800 gcggcctcga cctcccaggc tcgagtgatc gtcccacctc agcctcccag gt #agctggaa   1860 ctacagatgt gtgccaccac acccagctaa atttttttgt atattttata ga #gaagggtt   1920 tcattatgtt gctgaggctg gtctttaact cctgggttca agcaatccac cc #accttgac   1980 ctcgcaaagt attgggatta caggcatgag ccaccatgcc tggcccttaa at #gcatcatt   2040 cttatacatt cctttataca tacaaagctc gaagataata ataataaaca tt #attcccac   2100 ggcttctgtt gaatccccaa ctcctagcct gtgcctaatc aagtgtgccc tc #aattcatt   2160 ccaatggtct ggaatttgaa gactaaagga atcaaagggt atctcttccc tt #tgttcctg   2220 ggaccatcaa gttgtgaaat gacctcttgg ctttctgatc tgggtccact ac #cacaaatg   2280 aaatagaaag gagttaacac atttgcatta actgatgatt cttaatagag gc #aataaaat   2340 gagaaacccc catttcctaa tttaaatgtg gctttgggga taggataagt at #cctggccg   2400 ctccttagct aatcaggctc cagaagggac attaattgca gccagcgcat cc #tgctgggg   2460 agacccaagg ctgccagctc ccttgctgag aatggaatag aattttaatt gg #ctgtgaaa   2520 gcatcatggt aggaggtaca gggaagagtt ggtctgctcc cccaaagcag gt #tagggaag   2580 ctccaaggga aaggactcaa acaggtgcat ggcagcaaga acctcccatg ct #caggggcc   2640 ttcccagctg caacaccaca gttccaattc cagggctgga ctccagccta gg #tgaaggac   2700 cttctcacac ttgggcgatt agtactggag cacggagcct gtgaatcttc tc #atctctgt   2760 gcttccccca acttccatga gagaagcacc acatagttac tgtcacttgt at #tgtcaatc   2820 ttcgacttca gagaggtctc caaatataag ctcaactccc cagtgtactg tg #tgtgtgcc   2880 attagtccat gtgtatggtc tgaagcaatg gcacagcatt cctgtactgt tg #tttcagag   2940 caatgtgaat ttatttcact cacaataaat taatctaact atgcttaggg ca #aagttgtt   3000 ctcatggtaa tgagtatgtc ctttctgcca tggcaatttc tggaacacac at #atagacaa   3060 gaatgacatg tgaaggtcaa tagatgagac tatgaaaaac aagacaatat ag #ctttttag   3120 cataaatgta caatgatgca tgtggttttt ggagattgtt ggacaaatta ct #taacctct   3180 tggtgcctta gttttctaat tattaaaatg agggcaacag tattgctgat ct #aataaggc   3240 ttttggaaag aataatgaga tgtaaagagc ttagaatagt gcagagtctt aa #taaacatg   3300 tcttcgtgat tccttctagt gaagtgacat agagaagtgg gaccagtcat tg #acccaccc   3360 aagctctgtc aacctagtgg tatagtaaat ctgttggcaa aaagcaaagc ag #aacaacaa   3420 aaaaacgcaa gcaaaatcat tgctttcagg aaactatttt gtgagacaga ag #cttgataa   3480 aaattcaaaa tttgcagtaa actctacttc attaatactt taaaactcac ag #caataaac   3540 cattacttag aacaagtaag cagttttgtt tttattgtgc attttgatat tt #attttcaa   3600 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa          #                   #          3629 <210> SEQ ID NO 20 <211> LENGTH: 1144 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 tcgacccacg cgtccgcgct tgacttgctt ccagacaaag gttgtctcaa gt #ttgttgct     60 caaaccgagt tctggagaac gccatcagct cgctgcttaa aattaaacca ca #ggttccat    120 tatgggtcga cttgatggga aagtcatcat cctgacggcc gctgctcagg gg #attggcca    180 agcagctgcc ttagcttttg caagagaagg tgccaaagtc atagccacag ac #attaatga    240 gtccaaactt caggaactgg aaaagtaccc gggtattcaa actcgtgtcc tt #gatgtcac    300 aaagaagaaa caaattgatc agtttgccaa tgaagttgag agacttgatg tt #ctctttaa    360 tgttgctggt tttgtccatc atggaactgt cctggattgt gaggagaaag ac #tgggactt    420 ctcgatgaat ctcaatgtgc gcaatgtacc tgatgatcaa ggcattcctt cc #taaaatgc    480 ttgctcagaa atctggcaat attatcaaca tgtcttctgt ggcttccagc gt #caaaggag    540 ttgtgaacag atgtgtgtac agcacaacca aggcagccgt gattggcctc ac #aaaatctg    600 tggctgcaga tttcatccag cagggcatca ggtgcaactg tgtgtgccca gg #aacagttg    660 atacgccatc tctacaagaa agaatacaag ccagaggaaa tcctgaagag gc #acggaatg    720 atttcctgaa gagacaaaag acgggaagat tcgcaactgc agaagaaata gc #catgctct    780 gcgtgtattt ggcttctgat gaatctgctt atgtaactgg taaccctgtc at #cattgatg    840 gaggctggag cttgtgattt taggatctcc atggtgggaa ggaaggcagg cc #cttcctat    900 ccacagtgaa cctggttacg aagaaaactc accaatcatc tccttcctgt ta #atcacatg    960 ttaatgaaaa taagctcttt ttaatgatgt cactgtttgc aagagtctga tt #ctttaagt   1020 atattaatct ctttgtaatc tcttctgaaa tcattgtaaa gaaataaaaa ta #ttgaactc   1080 atagcaggaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa #aaaaaact   1140 cgag                  #                   #                   #           1144 <210> SEQ ID NO 21 <211> LENGTH: 1443 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 ggaaccattg gcctatattg ggttggatct attattatga gtgttgttgt tt #ttgtgcca     60 ggaaacattg tagggaagta tggaacacga atttgccctg cttttttctt aa #gcatacca    120 tatacttgtc ttcctgtctg ggctggtttc agaatctata atcagccatc ag #aaaattat    180 aattacccct caaaggttat tcaagaagcc caagcgaaag acctgctgag aa #gaccattt    240 gatttaatgt tggttgtgtg tctcctcctg gcaactggat tttgcctgtt ca #gaggtttg    300 attgctttgg attgcccatc tgagctctgc cgattatata cgcaatttca ag #agccctat    360 ctaaaggatc ctgctgctta tcctaaaatt cagatgctgg catatatgtt ct #attctgtt    420 ccttactttg tgactgcact gtatggctta gtggttcctg gatgttcctg ga #tgcctgac    480 atcacattga tacatgctgg aggtctggct caggctcagt tttctcacat tg #gtgcatct    540 cttcatgcta gaactgctta tgtctacaga gtccctgaag aagcaaaaat cc #ttttttta    600 gcattaaaca tagcatatgg agttcttcct cagctcttgg cctatcgttg ta #tctacaaa    660 ccagagttct tcataaaaac aaaggcagaa gaaaaagtgg aataaaaata tt #acttcatg    720 ttcctccttt ctaaattact aacttttgtt atactggtac tgatattttg tc #ccatttca    780 ctctcttctc atacgtgagt acttaagaat atgtacattc ttgctctgca ct #gtatgtgt    840 gagctatatg gtattgtgta aatttttttt gaaggaaaat ggaaattctt ga #gaaacagt    900 ttgtttaaag aaatatattc aaaatcattt gtgaataaac ttgatcatcc at #ctcaatat    960 tgtttgacat ataaaataat tataagtgta aaaaattaca atttagtgcc aa #cagtagtg   1020 agcatgaaat gaaactattc aaaagagaat atggcctgtg catattaaaa aa #ttcaaaac   1080 agtgaatgca gactggagga gtaacttttg caaataagat gaatatgctt ca #ttattaaa   1140 ctcaatataa aaggcaaatc atcagaatat ttttaaatgt tgtttgaaaa at #gttttccc   1200 aaggaaagtt tattatttgc tgctgtttca agaaaattac ttttactaaa tt #tttttgtg   1260 tgaatttaaa cagctaaata gggatcagta actttatctc tatccttaat ga #acatttgt   1320 tttattggtg gctggaaata tttctattgt atttctgtgt atatttttaa ta #aaattatt   1380 tttggcctct taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa #aaaaactc   1440 gag                   #                   #                   #           1443 <210> SEQ ID NO 22 <211> LENGTH: 1053 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 cttcgctggt gggaagaagc cgagatggcg gcagccagcg ctggggcaac cc #ggctgctc     60 ctgctcttgc tgatggcggt agcagcgccc agtcgagccc ggggcagcgg ct #gccgggcc    120 gggactggtg cgcgaggggc tggggcggaa ggtcgagagg gcgagkcccc tg #tgtcctca    180 gccatcccaa gaagggtttg ctggtccctc ctttcccccc gtcccacgag gc #cacctggg    240 ccagcccctt gtcctctgcc ttctgctggc agaggagcag ctggactggg gc #ctttggca    300 cagcagccgg tgtctcctgc gcccgcctcc cccatggccc catgcagccc ca #ggggcttc    360 ccccctgccc atggagtaga gcccgagatc ctggccacta tgccagttct ga #cctcgcat    420 ccccctaccc cgagcccatg cagtctggga acatgccgcc ttctctccag cc #tctgtgcc    480 tttgttccag gtggtctcac cctcctgtcc ctggctgggc taggtggtcc tg #tccaggct    540 cctgcagcgc ccccctcact ttgacactgg actaggatgc agcctccctt ct #gtgtcccc    600 ttgagggtac cctgggtccc ctcatcaggg gcagaggcat gaaagagtcg gg #gctggatg    660 gccaggggct tctgggcccg acgcctagtg cagcccctgg ggtcgtggtt tg #acatttgt    720 ctgcctggtg caaacaagga atccttgcct ttaaggtgac aggccctcca ca #ggcttcca    780 gacttgaagg aaaaggttta agaaagaaaa caaaaccaac agttagtggc cg #gtgagggc    840 ccaggctggt cagcgtcccg tcttgcacac ccaggggcct ccctttctgc tg #gagtcccc    900 tgtgtcctcc accacccccc gccgcccagc atcctacctg gactgcggtg ct #acgagggc    960 ctgcgggcct ttgctgtgtg ccaccctccc tgtaagtcta tttaaaaaca tc #gacgatac   1020 attgaaaaaa aaaaaaaaaa aaagggcggc cac        #                   #       1053 <210> SEQ ID NO 23 <211> LENGTH: 741 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 gtagacttct ggaccttgra tgtcactgag tttaagagaa aggtagtccc ag #gactctgg     60 tctagcagct gagtaaggct caggccttag aatggcttag cttgctccat tg #caatgcat    120 catcctggat gttaaaatcc agctgtctct ctgaaamcta aatatgaaag ac #tgagattt    180 agtcaacttt gctgagattt aatttacata taataaaatg aatcatttta ac #tgtgcagt    240 tcagtaagtt ttgaaaaatg tctatacaga ttcatgtaac tgccaccaaa at #tgagaaag    300 gacacttcca ccatctcaaa agattctgtg tgttcctttg tagtaagtct ct #tctacccc    360 atccctagac aacagctgat gtgctgtcat tgtacacata tattagcttt gc #ctgtccta    420 gaacttcatg ttaatgggaa gcatcctgta tgtactgttt tgtgtctggc tt #cttcagtg    480 tatttttgaa atttatccac attgttgtgt gtatccaaag tgtgttcttt tt #cattgcca    540 aataatgttc tgctatatga atatattaca aaatatttgt ctgtttatct at #tggtggat    600 ttttgcattc gttccagttt ggggctatta tgaataaagc ttctgtgaaa at #tcaaaaaa    660 aaaaaaaaaa aaaatgaccc tcgagggggg gcccggwacc caaaacggag ta #tttccctt    720 tttccccccc cccgcccccc g            #                   #                 741 <210> SEQ ID NO 24 <211> LENGTH: 946 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 ggcacgagcg aaagcctctc tcttaacaac ggtgccgcac agctttgccc tt #gaaagcat     60 ctctactgga ccggaacaca ctcatgtgcc ccgctccctg acccagccaa gg #ctgccctt    120 tcatctccaa ggctgagatg ttgccggtgg tcccatgaga gcctgcccat gg #gctcaggt    180 gcccctttac cttctgctgg atggacatct ggctgtgagc caggctgggg tc #atggccgg    240 ggtgagcgga ggcaggggtg gacggaggct tcgagggccc atcactagta gg #gtcattac    300 ctcttgccaa cagccggggg tgggagtctg ggtctcgctc aggccagagc tt #ctcaacct    360 ggagtccctg ggggtggctg ccaaaggtgt gtatgacaag cacgtatccc tg #gacatttc    420 cggggagagg tctggggctt tggtcacatt ctccaagggc tgctgggctt cg #gagcagtc    480 cccccccatg tctcagccac tacagggtcc ctctctctcc ttgcacccca ga #ccctccgc    540 tgccctggta atgagcagaa ggaaagtctt ggggtgtgct caaagtcagg ag #agcaaaat    600 atgccaggca aaagctcccg ggaaaagccg gaggagtctg gggtggccac cg #ggatgtgg    660 agcagcgagg gcaaagacgg tgaacacagc cctccagctg tctgagcctc ag #ttttctaa    720 tctgtagaat ggggatgatc atacctgcct cacaagaatg ttgagacaat tc #acagagac    780 gttctggagc ccctttcccc cgagaccggc attcatgagt ctgctgggac ca #gaaaaccc    840 atctcagggg cccagcgggg cacccaggag agtctggcgg tgcaagcgct gt #ataaacca    900 caagcgttct ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa    #                946 <210> SEQ ID NO 25 <211> LENGTH: 831 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (5) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (10) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (11) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (15) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (27) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 25 catcnacggn naccnctact ataggtnaag ctggtacgcc tgcaggtacc gg #tccggaat     60 tcccgggtcg acccacgcgt ccgggggaaw tcccagtcga tttttccaag ca #gtactccg    120 cttcctggat gtgtttgtct ctcttggctg cactggcctg ctctgctgga ga #cacatggg    180 cttcagaagt tggcccagtt ctgagtaaaa gttctccaag actgataaca ac #ctgggaga    240 aagttccagt tggtaccaat ggaggagtta cagtggtggg ccttgtctcc ag #tctccttg    300 gtggtacctt tgtgggcatt gcatacttcc tcacacagct gatttttgtg aa #tgatttag    360 acatttctgc cccgcagtgg ccaattattg catttggtgg tttagctgga tt #actaggat    420 caattgtgga ctcatactta ggggctacaa tgcagtatac tgggttggat ga #aagcactg    480 gcatggtggt caacagccca acaaataakg caaggcacat agcagggaaa cc #cattcttg    540 ataacaacgc agtgaatctg ttttcttctg ttcttattgc cctcttgctc cc #aactgctg    600 cttggggttt ttggcccagg gggtgaactt tatttcattt ccmcaggttg aa #actgaatg    660 ggcagttcat gktaaaatcm cttttcatgg aaagagctct atgtaacagc at #aataaaac    720 tgsctaccta gcagcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa #aaagggcg    780 gccgctctag aggatccaag cttacgtacg cgtgcatgcg acatcatagc t  #            831 <210> SEQ ID NO 26 <211> LENGTH: 1294 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 26 ctgcagaatt cggcacgagg ttatttcacc tctcttggcc tcagtttctc tg #tgaaatca     60 ggaattaaca tggtctctga gacccccttc tgatggtgaa tgtgtggttt gg #tgattttg    120 tggccctgca tcatgacctt atttagttct ctttcaacag gggatgtttt ac #tgccttgt    180 aaaatcctcg tgggactgcg tgtctttata ggagccaggg tgtaaatgaa ca #gaattcag    240 attggttcta atatatttta cctctaaaag aaagggcatg gggaggccat ga #ccttaaag    300 caggtttttt ctgttgtctg tgaagcctgt gatgattgag agtggctggg ac #tggcggga    360 cgatgtttgg gtggaagagg gaggccatct cgatgcgccc cgtcccgggg ag #gcacccag    420 cctgtaagga ggtgatgtct atctacactg agcgcaagga ccctgaaccg gg #ggaggctg    480 aggcggggcc tcttgattcc caccctgtcc cccagtggct aggctagtgt gg #cccgggaa    540 atgacttcca tctctccctc caggcatatt taataagagg ccagtatttt ca #gattctgc    600 cgcttctgga cgaatgtctc agagagctgg gaggcgccct ggaggatgga ac #ccttcctt    660 gagcgttgtt gaggtgtgtc gggggtgccg tggcacaggc cccctcccct gg #ggggcatc    720 actgttccct tgctctgcat ccccgctgtt tcccctgccc ctgaacaggc gt #ggagatgt    780 gcacgggaca ctcggaggcc ggatgctcaa cagagtggag tgccgcgacg gt #gtggccgc    840 agcctggctg tgccttcacg acgcagctgc aatcagagga gctgtgggac gc #tgtcccat    900 gtggacacag cccactcact gggtgctgct cctgtgctgg gcgctgcact tt #tattgtcg    960 ttaaaaattt atattaagat gcggccgggc atggtggctc atgcttgtaa tc #ccagcacc   1020 ttgggaggcc gagacgggcg gatcacgagg tcaggagatc gagaccatcc tg #gcttacat   1080 ggtgaaaccc cgtctctact aaaaatacaa aaaaattagc cgggtgtagt gg #tgagtgcc   1140 tgtagtccca gctactcggg aggctgaggc aggagaatgg cgtgaacccg gg #aggcggag   1200 cttgcagtga gccgagatcg tgccactgct ctccagcctg ggcgactgag cg #aaactccg   1260 tctcaaaaaa aaaaaaaaaa aaaaaaaact cgag        #                   #      1294 <210> SEQ ID NO 27 <211> LENGTH: 1656 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 ggcacgaggt tcacagcacc tgatttgcaa ggcagctata caagttcctg ga #ctcttgta     60 gttccggagt gtttcacctg accttaagcc caccccatcc atctttaatc aa #gaaaccat    120 gtgctttccc gcatgcctgt gttcccccct cacgtgtctg ctgtctgtgt gg #aagcctgg    180 cctggcgcat gctgtggtgc actgcatgct ggaacccgtg gagtttgcac gc #gtggtaca    240 gtatgaggcg ggtcacgttt tgtagtgtgt gccgtgggct cccgagaaac aa #gttaaagt    300 gtgtgctgaa atagatttta ttgacataaa ataagcctta ttgctaaatt ta #agagaatg    360 tgttacaaat gttttttgct aaacatcagt attgattatt ctacatgatg ta #cttattga    420 cataacaacc tgaaattctt gattttagac aatttctcct caagttgatt ca #gctgcatg    480 actctcagaa atcagtcatt ttttattgta gattgctggt tttcttcctc ta #gtttgtat    540 cgtgtatttt cctcctgtgg agaaaatgtg gttggcaaga aatgccatat tt #taaagctg    600 tatcgtggct gttaatgcag aaaacaccag tgtactgcag gctgtttggc ag #tggggctg    660 gggctgagtg tcctgccctc agtggcctgt gtctgtgctc ttgttcgctg ac #atgcagat    720 acaggggcag atctgagggt ttgatggagt gcagaaggcc acacgtgtgg ct #ttctgtaa    780 atgcagaaac atggaatcct tgagcagaca cttgtcttct ggagcacctt gc #atggattt    840 cgcctcctga tgcttcattg ccgttaatag agtggtggtg gttgtgttat ga #gaaatttt    900 gtctaacctg gcttctgaaa tttctcaaac taaatattca tgctgttttg tg #tttttctt    960 aatgactgag gctagtgata ttactcagaa aagtaacagt aacttgggtc tt #ctgagcgt   1020 caggatgttc accatttaac ttgtttctcg ttagtgtcta gtacgtcggc tt #tcggtagt   1080 gtaggtgtgt gttctgtgtc ctttcccgtg tgtgcctgca ctagtggcag cc #tctgcttc   1140 caggtcagtt tagagtagac tggctctggt attgctagca agtagttgct gt #tacccagt   1200 gtagccatga agcccagctc cttggatctt gacatatatg ttccaggcaa ag #tacgtaat   1260 ccagacgttt ctaactcttt ctagatgatt gcaattgttc tccatgttgt ct #gttaggcg   1320 ttatgttaat tctcgatcta acagtgtgcc tgtaacatat atggtagtga ag #agacatca   1380 catgcagaga ccgttttcct tttatcaact acaggtccgc tatcgacgag ag #cacctttc   1440 tgctaggcag tcaccctact tcccgttgtt ggaggatttg atgagagacg gc #agtgatgg   1500 tgctgctctc ttagctgtga ttcactatta ttgcccagag cagatgaaac tg #gatggtga   1560 gtggagaatg cttcctgaaa cagatccgaa aaggcttaaa ggaaaattat ag #tgtacatt   1620 gatccacata tatattaaaa aaaaaaaaaa aaaaaa       #                   #     1656 <210> SEQ ID NO 28 <211> LENGTH: 1350 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 28 gtgacttcta tattcaatag atttttgtaa atgttaaaac atctatattt aa #atgttaaa     60 acactaaata tagagagggg ctttatttca atcatagagc aacaacaaaa at #aatgctta    120 tagctaaact gcctgttcta gaaagcatct gctttttcat gttattccta aa #tcctcttg    180 tcatactttt gtcattgaac aatgctctcc ctctcgtctt ccatcctcat tc #agaatttt    240 tagaagacca caatcgtgga gatacactac ccagtattgt ttgatacatt tt #tatttgat    300 aaacattcag tgcaggaaac tgtgatttgc tatatgttta tgtatataat ct #tattctgt    360 agtcatcaga atgttaatgt aaggtacatt tgatttttat tttttacatg tg #tagttttc    420 tttcttcaca gtcaaagcat ttatattatt gggggtgggg gcagggaatt aa #gttggtgg    480 gctcgaaaat ccattcatat gtatctgtct acaaatgtct ggggataatt ta #aatttgaa    540 acctaagtta tatatagttt ggcaatgctc ttcttcaata tttacaataa ta #ggatgatc    600 tacaagaaaa taagtttctt tttgcaaatt tttatcatac taaagttgtt ct #tttaattt    660 agcatatcta aaataggatt tagttcagtt tagctcacac aggtgtttgc tg #acattcat    720 tggccattta atacagtgtt gagtggttct ccgtaaaagt ataagtgcta ac #actacgaa    780 gaaatgcaca cgatcattct tgctcacttc tataacaaac ttacataaaa tg #gatttaaa    840 aattcctact cacagcctaa aacttctgga gttcactacc tttttttcaa at #tcatagta    900 agatcacctg tgtattttat attttagtaa agccaattat gaagtacaag ta #tcatacac    960 gtacttttga gctactatta tttgaaaaaa atctgccaaa tagcatcttt ag #gatatatt   1020 tacattttca ctcatctaaa aagtatacaa aaataaaaag tggaaaaagg ta #tcttctga   1080 atgttcaaga gcatcctata gtgccaaata ataaagcacc atttttttct tc #ataaccag   1140 gattaaaatt catatatact gcagggcaga catacatatg atagcttgtg ct #gattaatt   1200 taaccccatt tgtaaacaga tgaaaatttt attttcttat ttcatttata ag #atggctca   1260 atgtattggg aggcttcttt tttattacag aaagtgtata ttggtatata at #aaatgaac   1320 ttttcaaatg aaaaaaaaaa aaaaaaaaaa          #                   #         1350 <210> SEQ ID NO 29 <211> LENGTH: 1766 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1743) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1748) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1749) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 29 gctcagcgcg ctgcccggct ggggacccgc gcacctgcag cgcccgctgc tc #ggccctgc     60 atcctgcctg ggcatcctgc gcccggccat gacggcgcac tcattcgccc tc #ccggtcat    120 catcttcacc acgttctggg gcctcgtcgg catcgcgggc cctggttcgt gc #cgaaggga    180 cccaaccgcg gagtgatcat caccatgctg gtcgccaccg ccgtctgctg tt #acctcttc    240 tggctcatcg ccatcctggc gcagctgaac cccctgttcg ggccccagct ga #agaatgag    300 accatctggt acgtgcgctt cctgtgggag tgacccgccg cccccgaccc ag #gtgcccag    360 ctctcggaat gactgtggct ccactgtccc tgacaacccc ttcgtccgga cc #ctccccca    420 cacaactatg tctggtcacc agctccctcc tgctggcacc cagagacccg ga #cccgcagg    480 cctgcctggt tcctggaagt cttcccagtc ttcccagcca gcccgggccc tg #gggagccc    540 tgggcacagc agcggccgag gggatgtcct gctccaatac ccgcactgct ct #ggagtttg    600 ccctctttcc caaggagatg ctgctgggga gctggtatgg gtggggtctt tc #cctttaca    660 gacggggcag atgccaggac tcagcccatc ctgaggagga cacgtgtcct ca #tggagagg    720 gtgctccggc ccaggcgggg gagtcggtgc ccagtcagca gctctgccac ca #tcctgctg    780 ggaactgggg gggcctctat tgggttatag gcaaggcctt ttctctggca tg #gaattgtt    840 aattttctga cacgtctaga tgtgaaattt ctgaaaatgt tgaagcagag aa #acattcac    900 acacaaaaag caacatagtc atgtgggtcc agatggcctc agtcctagat gt #tggcaccc    960 tttgctgtgt ctcctcagag tatcctgttc cgcctcctgc cacctggacc tc #cctcagtg   1020 gatgtcttcc ctcccccgac cccagcctgt cagtccgagc acagtgcagg tt #tggctctg   1080 acttgggcct ttggctgcag tgggggtgga tttcagagcc tctcatggca gc #atctaagt   1140 gaccagagct gggatgagag aggggaaggg gcaatgtgag tggcgctatg gg #acgggcca   1200 gccctgctcc tgagccagcc ccgccctctg ccccctggcc ctgggctctg tg #ctagggat   1260 ggtgaagaat gggggcgtgc carcctgcag gagtgggaag caacacgcag gg #gtcccgga   1320 cctctcmagc cttgccctya cgcttatccg agctcccagt gtggttagca ca #gagctcac   1380 ccaccttgcc tggctcccag ctggggcctg tcctcactgg tgctccaggg ga #agaaacga   1440 cagcctcact tctgtatgga ctgctgatgt ggcctgccat cctgttcagc gg #gcattgtc   1500 tttggagcag caggagacta ggatgcctct cactcacatg ccagttcctg gc #tggccagc   1560 tgctcagggc tcaggctggg gcctcccatt gacatcctcc ccctacactc cc #tctctgag   1620 cctccgtcgc ccctcctgtt gggtaagggt gttgagtgtg acttgtgctg aa #aacctggt   1680 tcatatataa taaataatgg tgatgaaaaa aaaaaaaaaa aaaaaaaaaa aa #aaaaaaaa   1740 aanaaaanna aaaaaaaaaa aaaaaa           #                   #            1766 <210> SEQ ID NO 30 <211> LENGTH: 2790 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 30 ccgtcttggg ctactggcag ctcctctcct tgggctcctg gctgccaggc gt #tggtgcca     60 cttcttaaag gcctggaacc agggaggaga ggaaatgcta ttgttgtggg ct #ttctccgg    120 ggtctgtgct gtgcctgcta gagcaacccc tgtacccagc tccttttgtc cc #cagggccc    180 ctccctctgc cccaagcagc cagccagtct tgcctaggcc aaatgcacaa gc #tcagaata    240 gatctgatgg tgagctggga agctgtactc agagcagagc aaatgaggga gg #gggcgctc    300 aggacccagg ccctccatgg gctaatgtga gtggcagcca tgcctcatgc ca #caccttct    360 tcgcaaactg atggaccggg tgggcctggc ctgagctggg gccacaaatc aa #atcaaggg    420 ctccagcatc cagcctgtgt gttctgtaat ggaactgacc ccctcccctg aa #aacgaaag    480 ggccccgggg ctggcaatca gggaaagctc cacggtgcgc ggctgtggca ca #aacttctg    540 gaaggctggc tgactggaat gcagggaaaa cggcagtacc tgggaaagga cc #cacccatc    600 ttcctgctgc tgtaactgct gagccactcg cagtcccagg atccgctgcc ac #cacgtctg    660 ccaggcccat ctcaggtgcc actccctgag ctttggggac agttggcaga ga #aggcctct    720 tgtgctcacg ctcccccgca gtccccagcc cttctgcctt tctcccccga ca #ctgctgca    780 ccagagtgaa agggctatgg caagggggtg tcatctgagg agtattaaga at #gcagattc    840 ctgggcctgt cccccaaggt tttggagtca gtaggtccaa gggccatact tt #tgagaggg    900 gtttgggtta agtatgaggt gaaatgggag atggtcagtg tggagagggg tg #cacccact    960 caccagggtc cgcaccagct gctctgcccc ttgggcatcc acccagtgct gc #catgccac   1020 tgccaggcac ctggcctgct gggaaccccg cagcccgtga agcagtgcct cg #aggcaccg   1080 gcgctgcagg tacttcctcc tgatggccaa gagcatcgtg acccttcagg gc #cagaagga   1140 gggcagagcc atgggcctgg gcctgctttt ccaggatcct gcaggaacga gc #actggcca   1200 gagagggccc agctgtagcc atggctcagg caagcccctc agcccttgcc cc #catccctc   1260 ggacccacca aactgcacac acagctcctc ttaccgtagc ctccgtttat gg #gccttgct   1320 ttgggctttg caggctctgg gctcagggct ggagtgcgct cttggtccct gg #tccctcgt   1380 ccacaggggc aggcctggga cccagctact ctgtccaggc cactgtggcc ag #agctggaa   1440 ggcagggcag agggaatgtt ccctgcaccc tggaaagggg agttgagtca ca #agaggtta   1500 aggtgggtcc aggaaggcag ctgctcttag tgcccgccta ggagttgagt ac #agtgagga   1560 gggtggagga aggtgctgag cttagccttg tgccctgccc ccatctcccc ag #gcctccag   1620 cctctcccgg ctgcctgcca cccaaagaga aatcacaggg gcggggcagg aa #tgcaaagt   1680 gttttctcag aacagctgaa acattccgaa gagggaatgg atggggagaa tg #gtcaatac   1740 acataagacc gtgtcccaag gagctgattt ccaggcccct gaggactgga ga #ccgcttca   1800 cccctgcact tcagacaccg tttgtccccc ggggcaaggt ctccttactc tg #agcccagg   1860 ccgttcccct tggcttcctc cgtccaccca ggctgcactg cagtgatggc gc #gggaggca   1920 ccagctctgt ggcctgtgtc cagcagctgc gggtctgaag gaatagccag ag #aggagcac   1980 ctgaacccca tgggcttgga cttcctgggg ccccgctggg atttcttcgc tg #ctctagct   2040 ggcaggacac atcccggcct cttcttccac ccattccccc atgtggctga ag #acattcca   2100 acaatggggt gggcccataa tagttagccc tcagtcagtt cccggagcac ag #ccctggga   2160 gggggctatt tctctcccca ctgaaaacat ttcaaagctg agttacttgt ct #gaggcctc   2220 atccctcgga agccgtctga ctccagagtc tgagcccccg gctagtaccc ta #tagagagg   2280 gggctctcca aaggggctgc tggggcatgt gtgcctgtgg cagaaaagag ga #gaccctgg   2340 aattcagcac cctgggtgcc attcccagcg tttagtttct agaggcctca gt #ttctccat   2400 cagcttatgg gatccttgtc tttactgaca agaatggaat agaaatgtaa aa #gtactctg   2460 aaaagcaatt gccctgtaac ttatctagaa agaaaagacc ctgagactcc ag #aatctgct   2520 gttgccatag ccccatatgt gtgaattctg caactagcca aggctagttc ct #ttcaattc   2580 catttaaaaa acaaaaacca gcaggtgtgg tggctcatgg cgtaatgggc ct #gcccaatg   2640 ctttgggagg ccaaggcagg tagatcgctt gagcccagga gtttgagaca ag #ccctggca   2700 acatagtgag atcccatttc tacaaaaaaa aaaaaaaaag gaattcgata tc #aagcttat   2760 cgataccgtc gacctcgagg gggggcccgg          #                   #         2790 <210> SEQ ID NO 31 <211> LENGTH: 1417 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 31 tttttttttg attaaaaaaa tttaaaaaat tataaaatga tgtcctatat ga #gtttaata     60 catgacgttg gaggagcata gagatagacc tagactaggc atgtgtatgt gt #gtgtgtgc    120 atgtgtgtat gcatgcatgc ttatgcatgt gtgtgtgcat gcatgcttgt gt #gtgtgtgt    180 gtgtgtgtgt gtagagcctt ggtcatcccg acagagcaaa gacacaggag gg #tggcacat    240 ggaagaacaa gtgactccac cctcccttgc acagttaaaa tctggccaag tg #agagggga    300 gatgggagag gggagagggg agaaaggaga agaggcactg actggagggg ct #gaagcttt    360 gtccctcctg ggcaggcgtt ctccatccac acccctcttc ttggatagag ag #gataagca    420 ggccaaagat gcacgaaacc tgagttccac tgtagctcca gacttctaga aa #agtcaaca    480 gcccctgtat ctctagctga tcctctgttg ttcaatgtct gcattaccgc ac #tgggagac    540 acttgacaga ttgggcctgc cgcaggccat agcagacatt gggcagccct ag #aacgaagc    600 tgactgtcct tggaatgtgc cacaggggtg tgacgccccg gccaactcca gt #gctgccta    660 aaatggcctc ttgcaacatt cccctctctt catcttaaat cagggacttg aa #gccacaaa    720 atggcaaata cacagttctg gcagtcgttt tgagtattgg agaaatcgct ct #ggccatct    780 gttttgtctc cagcatgttt ctcacggaat atccacggat atatccatgg at #ataacaga    840 catcctgcca aggcagagct tggctcttga gaactcggca agctcagtgc tt #gcctggat    900 tcctgcctca tgtcccatcc agtgtttgga gaaaagctct gagagaaaga tg #aatgtctg    960 aggccacaca gcctagaagt agtcaagagc acaggctcta gaactagccc ca #cgtgggct   1020 gaaatcccag caccagcgcc tgccggctgt gtgatgtagg agagcttctt ac #cagctctg   1080 tgcctcactt gtctcacttg taaaatgaga ataagaattg gccgggctcg gt #ggctcacg   1140 cttgtaattc cagcacttcg ggaggctgag gtgggcggat cacttgaagt ca #ggagttca   1200 agaccagtct ggccaacgtg gtggaaaccc cgtctctgcc aaaaatacaa aa #attagcca   1260 ggcgtggtgg cgggcacctg cagtctcagc tactcaaaag gctgaagcag ga #gaatcgct   1320 tgaacctggg aggtggaggc tgtcagtgag ccaagatcac accactgcac tg #cagcctgg   1380 gtgacagagc aagactctgt ctcaaaaaaa aaaaagg       #                   #    1417 <210> SEQ ID NO 32 <211> LENGTH: 1906 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (617) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (940) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1461) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1901) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 32 tctggagctc caccgcggtg gcggccgctc tagaactagt ggatcccccg gg #ctgcagga     60 attcggcacg agcccagctt ctggtggttg ctggcaatct ttggtgttcc tt #ggcttgta    120 gatgcatcat cccaatctct gccttcattt tcatgctgca ttctccctgt gt #gtgcatgg    180 ctgtctctgt gtccaatttt tcccttttta taaggacacc artcatattg ga #ttagagcc    240 caccctaatg acctcatctt aacttgataa tctgaaagca ccctatttcc aa #atagggtg    300 acattcarag ggtgttagga cttcaatatc ttttgagggg acacagttca ac #ccataaca    360 cctaccaacg gtttctggaa tcatctctca aaataaacta tctaaactct aa #tctttgwt    420 tcagggtcag cttctagcag aactcaatgt aagacaccct tttaaagatg gt #acctcaga    480 ggtacagaca gattgtgaac cttccctagc acagtgtaac aagtcccttg ca #aaaatcct    540 gatttgagtc aacattgtaa tttcttgctt aaatctaaga atatgcctyc ca #gcttcttc    600 caagactatc tgggggnagt tgkttctagg gtggctcaat ttattcgttt tg #aacgctga    660 tggccgtgca ccacaccgtg gctcaatggg tgttgaggac acgttcacat gc #aataaatg    720 cacagggctt tgccattagg tggcattagg ggaaggacac ttycagcata ct #gcagaggc    780 attctcttcc agcctggttg cctgtcaagc acctgctgag atgactggcc ca #gacggaga    840 ttgtggggtt gctgagccag ccctgtcccc tccttatctc cggaagatgg aa #taaatgcg    900 tgtcagaagg gagggtgcct cccatctcgg grgcaccggn gttgcccttc ag #aaaaacat    960 tgcctgcaca ttttgtagtc ttgaaatgaa tctgagtggc aattcaagcg gg #cagagctt   1020 gttttggatt ttagacagtt ctacctgcgt gctcctcttc tctgctccag ct #ctgacatc   1080 tcggctccac atacagtggt ctgaagtggc atacggaccc tgagaagagg ag #aggcaaag   1140 gwrgcagctg tggctggccc ctcctgccyt ctggtctcct tggctggtga gg #gaagaaca   1200 aacacagttg tgtttccagg tcacagctgc cagggctcca cctgtggggt gg #gtggctcc   1260 agttctgttc tgagttaaga aatgctgcaa atacgttctc cttraaagag mc #ctaggaat   1320 tgccatttct ttctgcagct ttctgtagcc aataagcatt tgaggaactg ra #gaagggtt   1380 cagccctgaa ttgcaaggga aaactgtgtg agtgtgtttt agttaagaaa aa #agttaatt   1440 ctagtgagac ctgcttgttt ncaaaacaga tgtataggcc agacagatgt ac #agggatga   1500 ccttgacttt cttttgtcat tgcaggaagg tggggtatgt atgacccctg gt #taagacca   1560 ataggaggcc gggcgcagtg gcttacgcct gtggtcccag cactttggga gg #ccggggcg   1620 ggtggatcgc ccgaggtcgg gagtttgaga tcagcctggc caacatggag aa #accccgtc   1680 tctactgaaa aaaaaaaata cagaattggc cgggtgtggt ggcatgcctg tg #gtcccggc   1740 tgctcgggag gctggggcag gagaatggct tgaacccggg aggcggaggt tg #tggtgagc   1800 cgaggtcgcg ccattgcact ccagcctggg caacaggtgc ggaactcggt ca #aaaaaaaa   1860 aaaaaaaaaa raactcgggg ggggmaccga acccgggtcg nacatt    #               1906 <210> SEQ ID NO 33 <211> LENGTH: 543 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (367) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (376) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 33 ggcacgagaa aatattgact cctatctggc cttcatcaac tgacctcgaa aa #gcctcatg     60 agatgctttt tcttaatgtg attttgttca gcctcactgt ttttacctta at #ttcaactg    120 cccacacact tgaccgtgca gtcaggagtg actggcttct ccttgtcctc at #ttatgcat    180 gtttggagga gctgattcct gaactcatat ttaatctcta ctgccaggga aa #tgctacat    240 tatttttcta attggaagta taattagagt gatgttggta gggtagaaaa ag #agggagtc    300 acttgatgct ttcaggttaa tcagagctat gggtgctaca ggcttgtctt tc #taagtgac    360 atattcntat ctaatnctca gatcaggttt tgaaagcttt gggggtcttt tt #agatttta    420 atccctactt tctttatggt acaaatatgt acaaaagaaa aaggtcttat at #tcttttac    480 acaaatttat aaataaattt tgaactcctt ctgtttaaaa aaaaaaaaaa aa #aaaaaaaa    540 aaa                   #                   #                   #            543 <210> SEQ ID NO 34 <211> LENGTH: 1452 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (283) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (596) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (607) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1275) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1284) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 34 gaattcggca cgagattaag ttgtgcactt taattgggtg aattgtacat gt #ragttata     60 tatctractg tagttgtwat taaaaaacaa caggaggcca tgtgggctgc ta #ggagtagc    120 aatgtctgty cccagccagc aggtagagac cagggctgga cagagkagta tg #ggctgtgc    180 tgcagattat ttgtggtacc caactgttgc ataaaacagg gtgtgatctc tt #gcattgct    240 atgcatgagt ggattcccag taaattgtgc caggctgcct gantgatgtg tg #gcttgtgc    300 tttggatcgt aatgcttacc tatgctactt aagttacata ccctgtggcc tt #tgtggcca    360 ggactgtggg ctactacctg kagtgattcg ttaggggaaa ggacccacag cc #tgtgcagg    420 aggaaaaaag catctctgag tacagggtgg atgagctgga tgagctgccg gg #caagagcc    480 acgcacaccc aggtggtgag tcttaaggat aaggtggaat ttgccccata gc #tgtcctgg    540 acagaaactg cccagagaag aatgaatgga ggacataggg ctctgtggtc cc #accntttt    600 ttggganacc tgtgactggt cctgttacca tgtcaactta gccccaaacc ca #tctctgat    660 tgacttggtt gcttattttg gcacattctt gctccacaca gccacataca ta #ctggctgc    720 tcctcsaagg ccaggcagat gcagcagctg ttgggccagc aaagaggaar gt #cctggaag    780 gttctggcct gaacgctgca tctgttgtgt gacagccaca actgctcagg ct #tccttgtc    840 tgtgggtgca ctgtggggag gagtgttatg ataagaacat tggctctcag tc #tccctggg    900 gagaagtttg gcctcacgtg ggatttgggc gttgccttta ggaaggctct ct #gcatgtct    960 agttccagtt tgtactggga agaattaaaa aagtctgcca gcttctttag tt #tgtcctgt   1020 cttttgtgat gattctttct gagatcccct cctatcagct caggagtggg at #tttctgga   1080 gaaggaaagt gtttttctgt tcctcactgc tcaccttggg gcattcagga ac #atgggcct   1140 gatgaatttg cttgaaggca gtctgtaatc ccatcacttt gggagccaaa ga #rgcggatc   1200 atttgagctc aggagtttga gaccagcctg agcaacgtga caaaaccctg tc #tccaccaa   1260 acaacaacaa caacnacaac aacnacaaca acaactacaa caaactgggc tg #gatggcac   1320 gctcctgtaa tcccagctac ttgggaggct gagatgggag gattgcttga gc #ccaggagg   1380 tcaaggctgc agtgagccat gattgcacca ctgcattcca gcctgggtga ca #gagggaga   1440 ctgtttcaaa aa               #                   #                   #     1452 <210> SEQ ID NO 35 <211> LENGTH: 2908 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1653) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1655) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (2850) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 35 gagctctaga ctgatcgtag taagttttgt gacttaacaa tgaaagaaag ta #gaaagatg     60 ctttgggttt tcaaaatgtt gttttttaaa attgttcttt gggtgaattt ac #tcagtgct    120 gctttgagtt gtatacagaa acaaatgttg ggaattgctc ctcagaaatg tg #ttcctaag    180 ttgtgttttc aactttacat catgaggtga agcattaggg aaagagattc tt #tcgatttg    240 tttaataatc taattataca gaccaaagtg ctttacgttt ctgctctatg tt #aatgtttt    300 agaatggtga ttttgctgat taatttagac ctggcaattg aaataatgtg ct #cagaataa    360 taacatggtt atagttcttg tatgataaag tattcaattt cagaatagtg tt #ggaatcct    420 gagtttgaat aatgtgttgt atttagaaac atagccctat ctgttttaaa ca #aataattt    480 gttggccgat tgtccatggt tgagcatgac aaaaatacct cgtcgaaagg ca #agcttagg    540 taactgctgg caaaacactg ggtgcactat ttttctggat aaaatttata gt #tattttct    600 atattaccct tcaaaaggga tctcttcagg ttaaaaatca cgcttatgct ga #agtcttta    660 tctggtgtta actaaaaatc tcatatgggt tcataaccga ggtcactaat aa #ttcatttt    720 tatcacttgt aaaaatttgc tcaaaattcc aaaaaaatat tgatttgttt tt #tagtgatt    780 ttgcaggctg accccaacct aagttttgat aacatctggt aagtcagtat ag #ttctgtga    840 cttcatgttt taactaagaa ggaaaattca tagatattcc tattagattt ta #taaacctt    900 caaaagtctg aaacttaatt ttgagtctaa attttctgac actggcccct tt #taatattg    960 taagtttttg ttcactttct taagtaaaaa aaacatttaa ttactagtta gc #ccttaact   1020 gggaaactca ggtaatgaac tgctgacttt tctaaagttc tttaactgat ca #attctgta   1080 tagagggata tttatctaac cactttccgt attttacaag tgctctttct aa #aaaggaat   1140 aactattata gctctatttc cccaatctct ataggactca tgagagattg ct #tgtgtaaa   1200 tataaaagca ccatatgtgt tcttaactcc tatggctgct tgaagctcat ga #tgaaaaag   1260 tctttttgtc agttttaatt gttaagtaca gaacaaacaa ttgtttggtg at #ggcctggt   1320 tgaaagagag catataaata tatcccagtg gaactcacca aagaagacca ca #cctcagaa   1380 attattgcat tttctcatta tgtgttgggt ttgatttgct tttgttttta at #gcagctct   1440 tttaatataa agattcttga tacagtgaaa tctcttattt caagtgtaag tt #attcttca   1500 cccacccctt cccctgccat tgtatttccc atctgtttca aggagtttca ac #aatttaca   1560 ttgcatcgta tgcagtaggt actgcttttt cagaaagacc tggaaaacat ac #ctgctatg   1620 aatattttgt tcagatgtag ccatttacct ggntntcaag gttgccttct gt #ggagagga   1680 tcttagacaa aaatcttcct tgtatttact tgggttaagt gaagtccaaa tt #cttacagt   1740 atgctatttc aggatttctg atattaaaaa agaaaaaaca aaatctttat at #ctcttatt   1800 aacacttccc ccaagaaggg ttgtgctgtt atttattttc tattataaga aa #agttcatt   1860 ctttaagtag tttcttttac ctctaatcta atttcatacc aaatacctga tc #aatagaaa   1920 tgatatattt aagcagcaaa gattcctaat ccatcattat gaaaagtgtc ag #catactta   1980 gtagtgaaca gataaagtca atttgaatat aattccactt tgtttttaga ga #ctaaatta   2040 agattcaatt aacattatcc tatgaattct gaatgtgata atgtgattca aa #cagtcaaa   2100 ttttattaag ctcttagtaa ctcaggatag cattccatac taacctcaag tt #agcaaaac   2160 aaattagtta aacagcttgg ttcttagcag actgcttaaa agatcaagaa aa #ttttctca   2220 tcttttcttt ctacttagaa acattgcaag aaaccttgga cagtcttcac ca #gacctgcc   2280 atgattttat aagatttagg cctcagtgac atgctcctga aagtttcctg cc #agccatcc   2340 aaactaagca tccactcatt ccatcttccc aaagtcactc accgataaag gt #agcatcct   2400 taagttcatt tttgaaaggt ggaggaggat ctccccctgc ccaaaggaat tt #ttttatca   2460 gaataccttg aaaggggggt atataaattt ggaaaactta atttcttggc tg #tgtttgat   2520 aacagttcct atgcatggtt tttaatgtga ggtaaatttg tttctttctt ca #gaatacct   2580 ctctccaccc cccaccttat tcttcctctt taatgaatat ttttattgga gc #tcaaactc   2640 catgacttac gtgctcacta agttttcttt tttccccttg tttactctgt ct #gtatgtat   2700 gtcaaaagct ggcaaaacct ctaaaactgt caagaaaatg cttgaaagtt ga #tttgtcat   2760 agtgcaaatt catgataaaa tgtcttaatg ttatttggat atgtagtaca ta #gaaacaga   2820 aaaataaagt catttttata acttaaaatn aaaaaaaaaa aaaaaaaaaa aa #aaaaaaaa   2880 aaaaaaaaaa aaaaaaaaaa aaaaaaaa          #                   #           2908 <210> SEQ ID NO 36 <211> LENGTH: 953 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 36 aattcggcac gagaaccaag gtactttcag ctgcagactg accatacccc tg #tccacctg     60 gcgttgctgg atgagatcag cacctgccac cagctcctgc acccccaggt cc #tgcagctg    120 cttgttaagc tttttgagac tgagcactcc cagctggacg tgatggagca gc #ttgagttg    180 aagaagacac tgctggacag gatggttcac ctgctgagtc gaggttatgt ac #ttcctgtt    240 gtcagttaca tccgaaagtg tctggagaag ctggacactg acatttcact ca #ttcgctat    300 tttgtcactg aggtgctgga cgtcattgct cctccttata cctctgactt cg #tgcaactt    360 ttcctcccca tcctggagaa tgacagcatc gcaggtacca tcaaaacgga ag #gcgagcat    420 gaccctgtga cggagtttat agctcactgc aaatctaact tcatcatggt ga #actaattt    480 agagcatcct ccagagctga agcagaacat tccagaaccc gttgtggaaa aa #ccctttca    540 agaagctgtt ttaagaggct cgggcagcgt cttgaaaatg ggcaccgctg gg #aggaggtg    600 gatgacttct ttacaaagga aaatggtagc agcttcagtg agaaactgcc ct #tacaaaca    660 gtcccttctc tgctgtcaat ccaatactgc tcccaaatcc tgttttcagt gt #tcatttcc    720 ctcaaggcag gcgctgggct cccacgaccc ctcaggacag atctggccgt ca #gccgcggg    780 ccgctgggaa ctccactcgg ggaactcctt tccaaagctg acctcagttt tc #ttacaaga    840 acccagttag ctgatgtttt attgtaattg tcttaatttg ctaagaacaa gg #taataagt    900 aaatttttaa aaagcctttc tgctgggttg gattaaaaaa aaaaaaaaaa aa #a           953 <210> SEQ ID NO 37 <211> LENGTH: 3864 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 37 acccacgcgt ccgtttctga agcaatgtta atcctactag ccaagcatat ca #cttagtcc     60 ccactgtgag atgagggata tgtgcttaaa ttgtgaaaca aatatatgag tc #aggtattt    120 ttcctttgag tccaagtggt ttatgacttt ctttcctgtg ttctttgtat at #gtgggagt    180 tttataattt tttatcaaga atgaaaggtt ggcctgtgtt cttactggtg ca #ggctgtca    240 catttctctc tgttgcccag tcaggtgcta tggcatgtgc tgcttctggc gt #agtgtact    300 ctgtggatgt accagcatgt tcttcaaggt catgactgat tttccagacc tt #tggaattg    360 agataaatgt taaatttgta gctatctctg aatttcttcc agatactttt ct #tcatttgt    420 ttgtttgtag ggtaaacata cctgatagca gcaatttaag cataccctta ga #atgaccat    480 gtatggccag tgcacctgaa tgtgtgttcc aaggtaggga atccaggaat gg #ccaactcg    540 gagattcatt ccttactatg ataaatatct gagccccctg ctcatcctgt gg #aacatggg    600 cttattgggg attaaggccc tgagttttag gttaaatgaa ggttaccaga tg #gaggtcat    660 tagggggagg gtgttaaatg aaaatgcttt ataaactgca tgctgtttgc aa #gcagttgc    720 agttttcctg cccagcccgc agccactggc catgcagtca tgttgtccag cc #tgccgcca    780 ctggaccatt tctgtacata aggcagttct cctgtccgcc tgccaccagt tc #tccactct    840 ctccccatat gtaagcccct agtaaacccc atgtctcatt tgctgcctct gg #gtcttttc    900 ttcagcctct tgaacctagt gccttccctg ctgaggttaa taggggtaca gc #acaacagt    960 gttgtaacac agaaagtgat atttacaggg atatctctct cacaatatct ct #taggaaag   1020 gtaaataaaa tgttcacaac ttgtaggtga gtaattcctt agataagttg tt #tcttaact   1080 tgggaggagt ttgggaagga acctaagcag gctgcagagg ctgggcatgg ga #gcttgtca   1140 tggctggaag ttgaaatggt caactccagg cagatctcct ggggcaaagc ag #cctccacc   1200 accagtagcc cttcctttct gtctttcata ccccactgct ccatctgaag cc #tgaacccc   1260 ttccagaaaa ttgatggata gatttttttt ttcggctata tatagtttta ga #ggttagaa   1320 ctagatataa ttcagtctag aagatttctc cttccccaga aatgattgtt tt #tgtgcaaa   1380 gccccgccaa aatagtacgg agacttagac tgagttcact catcactaac aa #ttaacttt   1440 ataaacattc aacaagtagg acaactatta ttactgttac tcagaaccct tc #gctctgta   1500 tatacagttt gatttaagat gccacattta catggcattt tcaaccttca aa #ctctagca   1560 gattttaaaa ctaggtggat gaaaatagaa tcattctaat aaatgtagtg tg #tcagattt   1620 gaaaaatcat ttggtgagca ggatctctgt aaagttatat gggccacgta ta #caagacgt   1680 aactgaagaa aattaattca acagagcatg ccgtacttga acgacataga ga #tttactcg   1740 aactgaacta actcaagctg cagaactccg agcaagcctg gattgtaaaa gt #ctgggtga   1800 aaatagatgg agtatgccct gactgaacct ctgtactgcc ccacatgctt at #acaggtgg   1860 gggattggat ggctgttagg tgatcattgc attctctttt ggatccctat tg #agaagaaa   1920 tgataagaga gggaaaggat atggggcaag aacagtctga aaaagaaagg at #aaagttct   1980 cagactctct tcacactcta agaagaactt tctgaaaagc ttggattagg tc #tggcaatg   2040 gatataataa gcaaaggact cttggaatgt gttcttggct cttagcccca cc #tctgactt   2100 tgagcaaatc agctgatttc tctgcctgta aaataatagt ccctctgata tt #aatactta   2160 cctcatgagg ttatttagag gatagtgttg gtaataatgc cttgtgttta ca #tcattcct   2220 ttcacagaga gctcaaagca ctttacatgc attgagagag aagcttctcg tg #aagagtaa   2280 atagaagtgt tcactttttg gaaatgaact taggccataa gagcctgaat tt #aatgcatt   2340 gcaggaagaa atatggtaca tagtgaacca gtgggtcaac tgaatttttt gt #tccactaa   2400 gagtcccctc ctggctcctt gttttgtgaa ttgaggaata tggtgagtcc ct #acacctgg   2460 atgggaaatc ccacatatgc aattggaatg gtctctcacg acacatgcag ag #attgaaga   2520 acagtctgga cattttttga taacgttctt tgggccttgg tagtagctga aa #gacacctg   2580 agaaatctta gctcagagct acagaatgac actaatggat cccagaaata ga #aatgtaga   2640 tgtggagtgt tttatctgtt tatttcacct caattcaacc aatactcctt ga #gtgccttt   2700 tatatacatg attttgagtg atgtggagaa ttaaaagagc cccacatgct ca #ggaaagtt   2760 aaccctggtt ttagcaagga aaagaagtag gatttccaaa tagataagtg ca #ccgggtat   2820 gtggaagttc agaaaagctt cccagtattt cagcccatct acttggccat tc #tcaaccat   2880 gtattactca tgtaccaagc agtatgctgt tcacagagag atccaatctc tg #ccttaggg   2940 atccttgggg aaaacatgta caaagagata gttttagcac attctagtaa ag #gcagtgat   3000 caagggcacc tagccttacg tggcaattta gggaaggtac cttggaggat ga #gacttctc   3060 ctaaagtctt aagaattgaa aagaacatgg gaagggaatt ccaggctggg ag #agtagtat   3120 gttcatacgc cctcagtgtt taaccttctt tgaacaaaaa aatggccaac ta #cagaaagt   3180 ttggtcttat tgtagcctaa attgtactta ggggtacgag tgagaaacag gg #attaagat   3240 aaaggacctg ttttgctgtc ttgtttactg ttgaatagta gtatgaagta gg #tcctgaaa   3300 aactatgttt ttggggaaaa aaaaaaaaag actgaatgat atgttgggtt ta #agtccttg   3360 caggcaggct atccaggtaa ataaacatgg aaggtgatgg gaggtaatct gg #gctggaaa   3420 tacagatttg gaagtcaccc catatcagtg gtgtttaaaa tcaagagcaa at #gaaattgc   3480 acaaggagaa tatatagaat gaacaaatta ccatgggtga agccttgagt aa #tacagaca   3540 tttaagaagc aaacaaaaga caaggaaccc atgagggaga ctggaaagga aa #aaacagag   3600 aaataagaaa aaatgagagg agagaattga tacattttcc tcaggtgtgg ca #ttatggag   3660 ttcactggtg tctcatcaga gaagtttcag tccagtggcc agggcagaat ga #cattgtgt   3720 cttgttttaa agtaaatggg tagggtaaga aagttgagaa gggttagcac aa #acctctct   3780 ttcaagtcac ttgccttaga agagaaggaa gggtatggtt tctggggtgc aa #cccaggtt   3840 caagaagcaa aaaaaaaaaa aaaa           #                   #              3864 <210> SEQ ID NO 38 <211> LENGTH: 1411 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1395) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1397) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1401) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1408) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 38 ccggtccgga attcccgggt cgacccacgc gtccggcgtg aaccaccgtg cc #tggccgga     60 agtctttaaa aaataaagtg attctactct tctaagctta cagagaccag ac #caggtgaa    120 tgtaactggg gaaaatcaag atggtacctc tctgcattat cccgccagac ac #tgtatttt    180 atgcattcat gtctaggata cagtgtgaaa attaaaaagt ttagagggca ga #tgcaattg    240 tggcaagtga cctgccaata aagcaggtgc agctatagaa gctggcatag gt #atatcctt    300 aatggtgctt tctccctggg cttgtctttt tgttgttttt ttcccctata tt #cagagctc    360 cttgagaagt gataaacacc tccagctttc taacatcctc cccacaccat ct #caccatat    420 ccatctccca gcatccatct gcattcagct aagggcggga aactgaccta gt #gcctgtgt    480 tgcagaccat ttctgaggtc tccaccatcc aaggaggcac agccgtcatt ac #tgtcctcc    540 atgccttcag cagcccccct cacagctaag gtacatacca ccccttctgc cg #cgcctcca    600 cccctggcac caaggtcttc tgctgcttat gtctaaaggg atcacctata tt #taactgcc    660 tcagtgacct aacctctttc ttctcatgtg ccagatgtta agatgaagga gg #aatacmac    720 acatactcaa gcctcagcct gtttagttgt tttcactggg gctcgctttt ct #gggacggt    780 atttattatc agactggcaa gcctaactcc ataggtttac aggaagtagg ga #tattttta    840 taaaacaatt gtgtcctccc cacattttgc tatgttaata tttgcttcta ac #aatttgca    900 gctgtttcac tttttcctca tttgtctcta agttgaaggc tttgttggag gg #gacagagc    960 acaggaacag ccttgacagt ctgtaattat tgtacagata ttttaatagc at #ataaataa   1020 gtatattcct tttattttga aacaaaaatg atcagacact gccttttgtg tg #tttgctgc   1080 ctgtggcatc cttttttaaa aagactgtta catattaaaa tagtgtacat at #ataaatat   1140 tacctctttt gctgtacagt tgtgatagag actgaagatt ttattttttg tg #tgcttttt   1200 ataagaaaaa aattaataca ctaaagaatc ttgctgatgt gattgtaatg ta #cctatgta   1260 acttatttac ttttgaatgt tcttctgtat ctttaaacct tttattaaat aa #ggttttaa   1320 aaattcaaaa aaaaaaaaaa aaaaaaaaag ggsggccsct ytaraggatc ca #ascttgcg   1380 tacgcgtgca acganancag ngtcgagngg t         #                   #        1411 <210> SEQ ID NO 39 <211> LENGTH: 1182 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (496) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1162) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 39 ctaagctggt tcgcctgcag gtaccggtcc ggaattcccg ggtcgaccca cg #cgtccgat     60 aagcacccat gtctttgaat atgaatgtat ttgtaaaata ccacgtttca tg #tgtgaata    120 tgtgctttta ctgtacatag tgctattgtg caataggtct tatgctgttt tc #actcaatg    180 tgtgctaaga tctagcccca ttgactcttc tagaaatgca gtattgcttt ga #cctgccat    240 gtggcactcc acaatgtcaa ttgcagttta cacacattgc ctaaagtggg gg #acacctgg    300 gtgcccctga ccccttggca ccggatacag gccacgataa acatcctttc gt #gtgttccc    360 ttctgtgctt gtgtggcatg tgtacccagg atgggcctat aggtcacaga gg #tcagtttc    420 tctttggttt tccagatttt ctttagaacg gtgactgacc ctcctacttg ag #gccgcctt    480 ttctccttat ccttgncagc acttgtattg ccagactacc taatttttgc ca #gtctcatg    540 ggtagatagt ggtscagtgc tttamcatac attcatctga tcagcattaa tt #tggggaat    600 tttttcactt agcctttctg gtttcccttc ctgtgcattg cccattttct ca #tggagttt    660 cttatctttt ttggtttatt ctcaggagtt gcttgtacat tcttgggcaa tt #gcagataa    720 ttccaagaat gcatatttgg gctgggtatg gaggttcact ggtaatccca gc #actttggg    780 aggsccaggc agaaggatcg ctgcagccca ggagttcgag actagcctgg gc #aacatagc    840 gagacctcgt ctctacaaaa aaaaattaaa aagggggctt tgggaggcca ag #gcgggcag    900 atcatgaggg caggagattg agaccctcct ggccaacatg gtgaaacccc gt #ctctacta    960 aaatacaaaa aattagctgg gcatggtggc gcacacctgt agtcccagct ac #tctggagg   1020 ctgaggcagg ggaatcgctt aaacccagga ggcggagatt gcagtgagcc aa #ggttccac   1080 cactgcactc cagcctggcg acagagcaag gctccactca aamaaaaaaa aa #aaagggcg   1140 gccgctctag aggatccaag cntacgtacg cgtgcatgcg ac     #                   #1182 <210> SEQ ID NO 40 <211> LENGTH: 2457 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1622) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1713) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 40 gtcgacccac gcgtccgccc acgcgtccga ttataataaa aagtattatt ag #gacaggta     60 aactcatcca tctactgtga gctgctggct tggagaacga cttttgagga gc #cagtttcc    120 tgaggagaaa tgcttttata aagcactcat tgctttgtaa aaggagacaa aa #ctgatcct    180 aaatgaccac tccagggttg ctgattttgt ttctggctca tgtctgccta gt #aaaccacc    240 agcaagctgc tgaaccaggc tggaaacaac attgttgcaa ctgggaggga ca #tagagtac    300 tgtgaaagca gcgttccaac acatcttcac ttttacaaag ggataggcag ag #acttccaa    360 catagaggtt cttaaacttc gaggggttac aatccccttg caaatatttt ta #tagctatg    420 gaccctctcc tcagatctac agccaacatt ttcagtgcac cttaggaggt ca #ttttagtc    480 cacgcctctg aaaaagctgc actccaacgg ctgaagagca agccatacgg cc #gagaatgg    540 ggctcccttt gccttcatga aagctacttc ccccaacact aagactcagc tg #tacgtttg    600 cttagctcag tcacatttac attcttctgg gtgaactgta ccttttgagt ac #ctgccttc    660 attttctaga atcagaccta acaaggtcag tagaagcctg ggcagcagcg gg #cctggaaa    720 gacgaggcag ccagcatgaa ctgctgttct ctccctgacc acaaggcgtc gt #cttcctcc    780 agggtcaagt aattgttctt gttccgcctc acaggaatgt ggggaggaag ga #cgttaaca    840 ctataaaatg ctgcgtccta ccttaaactt gtactgtgag aaagctggaa ac #ttccacct    900 gtacagtggg tctggtttgc atgtttagtt tcatttgtgg gaactgcttg tc #caagagtg    960 agctcaggtc agggcagttt gtgcctatga gaattagctc agctatcagg ca #ggttttta   1020 gacacctttt taaaatgtgc tcgtgtttgg tttgttttgc ttactgtcag tc #ctgggtca   1080 atcaaaggtt tgtaagggtg agaattttta tgcactgcta tatcgcaagt gc #ttaaaaca   1140 gagactggcc cagatgaggc attcttcaaa tgtttgttga aatgaatgga ca #aactctta   1200 ggataaatcc taatttgttg gcaactgtta tttgatttta gaaggcaaac tg #attttatt   1260 ttagagaggg gaaggggagg ggaggctcat tagcctcttg gtagaaagag ga #ctatttct   1320 gcaaatgaat aggtttccac cttaagtagt gacagtcctt aacttcttat ta #tggagtga   1380 gtcttgaccg ctttccaagt tcaatagaag ttcaagattg cctctcagtg at #tagggaaa   1440 ttgaagcttt taaagctcct ggtctcagta attcctcaga ataaacctct tt #aaaaggga   1500 tattgatgga aatgtacaat taccagtaat tgaggtttta tctgagggga tg #gagatgat   1560 gaaatggttc cttcttggaa gttgttggca ttttggcttt atttttcaca aa #taaagtga   1620 anccatttaa aacgattgac aacgattata tagtgccatg tggaatacaa ta #gatattaa   1680 tttgtggttg gtttttctgc ctgctttaaa tgnaatgtat tatgtttctg gg #ttcctttt   1740 ttagctgtaa aaatacttcg tcactaaagc atgaaattta atcagcagtt gt #tcttcaag   1800 ttcctgaaag ctatarragt ttctcatgac ttgagtggtt ttttccctgc cc #accagagg   1860 agaaagccct tgtagaattc tgcagtgtta caagtgttcc ctacaaaaac tg #aaaccatc   1920 agctcctctt taacaagttg gctttttaaa agcacgtaat tacaatttaa tg #gtattctg   1980 taaagtggtg ctctaggcat aatttaaatt ctttttaatg actatatttc tt #caaaactt   2040 tgaaagaaaa atgtgttctt tttgctgcat cctttgtaag aagactgcca ac #agaggaaa   2100 aaggacttta caaattaaga ccatcttggt ttcatttcca caaagatgag aa #caaatcat   2160 ggtgttagga aaggatcctt agaagaacac aagaatttga aagcccttgg tg #gttatcac   2220 tactatattt catatttcca cagaagtgac ttagccaagc tctgcatttt ga #gcctgctg   2280 actttcattt aaaaggaatg aaaggctgaa aatccaggct gctgtgtctg ta #gataaagg   2340 tcaaaccatg tttgagttct tcactgttgt gtccacctaa ataaaactga gt #aagtaatg   2400 aaaaaaaaaa aaaaaaaact cgaggtcgac ggtatcgata agcttgatat cg #aaatt      2457 <210> SEQ ID NO 41 <211> LENGTH: 1847 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1279) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 41 gactccttag ctaagcatac aaggtaactg gccctgcctt gctgttttgc ct #cacctgct     60 aacgtgcaca tgctctgcca cgtgtacctg ctgctagtgg gacatgctga mt #tctcagtg    120 ggcctcatgg gccagaggaa gttgcgttgt tccataaact cagctctcag aa #gtgctgtt    180 tcctctgcct ggaattcgtc tatctgcttc aatagctgaa tgctgcttat cc #ctcaagac    240 tcaattctgg tggcacctca gttctgaccc ttccccagtc tctctttccc tt #ctccccac    300 aggctgtctc cccttgatat tttgcctgct tgctacccat gtccttttct cc #tggtgaca    360 gtgctacagc ctttccttgg ggaatcaacc ttgtctgaga gtggacagaa at #tatccacc    420 cctccatcca gggatgaacc aatgacctgg ccaatcacag tcgctgtgat tg #gtcctggg    480 acaggcagga gactcaagct aggccacaga gcatcagtgc tgagactgaa ac #tactggga    540 aaacaatatg ctctttctgc ctgaggcgcc tagtggatac aatggaagcc tt #gaggcagc    600 tgatcatctt tgccttgaga gaggtggctg cctaagaagg aagccaattg ag #aaaaagca    660 ggcagagaaa aaaaacagga acagatacaa caggcaagac tcagctgatt tc #tcatgtca    720 gcaaatacaa gagaaggaga ctggtcaaga tagagatgtc tgatgatact ga #gctcccga    780 ctccagccac tcctgaagtc attcctaagc ctctcgttac acaagccaat ac #atgatctt    840 gktagattaa aaatagtttg actttggtgt tgccacttgc cgtagaaagc at #ycacgatg    900 atacactcty ccgtgtgcty ccacacccta atttaaccty ctycatagca ct #gactcgga    960 ggccctatga gactgkgaac tctytgaggg caggaaatgt atcttattcg tc #tccaaggc   1020 cacgagactt ggtgtatgat agatattcaa tacaggtttg tgaaataaaa aa #tgaaggaa   1080 tgtttatcta gaaattaatg aagcttttca tttacttttt tacttcaggg cc #tttctgcc   1140 aagaactctt aagatgcctt aggatcttgg ttgcagccag gtggctgtcc tc #cccagagc   1200 ccttgttgtc agtcctctga agtcattggw cctggagata acagggaggg ca #ggtcccga   1260 ctgctgagaa agtccaggnc cccggatcct accattacca tgctacctgt tc #acttgggc   1320 ttcacccaag gccacactca tctccgtacc ccttcccaac agtggtgagg gg #caggagca   1380 cctggacatc aagaatcgag tgcatgcctg aacctgtcca ttacccatgc tt #ctgcagct   1440 ttgctcatgc tttcccctcc ctgaaacgct cttcccttgt ctacttaatt at #tcaaagct   1500 cagaggaaaa aaaatcacct gctccaaaac gtcttccccg aaccctgcag gg #aaccaaat   1560 tcaaaagcca tcagaaggcc gaggcgggcg gatcacttga ggttaggagt tc #gagatcgg   1620 cctggccaac atggtgaaac cctgtctcta ctaaaaatac aaaaattagc ca #ggcatggt   1680 ggcaggtgcc tgtaatccta gcaactctgg aggtgacgca ggagaactgt tt #gaacccag   1740 gaggcagagg ttgcagtgaa ctcagattgc accactgcac tctagcctgg ga #aacagagc   1800 aagactaggt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa actcgag    #              1847 <210> SEQ ID NO 42 <211> LENGTH: 2597 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42 ggcacgaggt tacacctcac cccctacttt cgctgactcc agaccccaga gg #ctgaggga     60 cagtagatat gctgcctgcc attgcctctt tccttgtgca tgtctgagtg gt #gccagcct    120 gatcagattc tgttgcagtt ccccgtgctt gccacaatgt cggtcgcgtt tc #ttatccaa    180 cgctgtttct gcttctggtg gtttgtgcta aatgcatttt caatcccaag tg #gtacagag    240 aagaaaagga ttgtctttaa aaagtggctt tgaatgtgtc gaaagcaagg tt #gcctgctg    300 tgtgtttttg acaattgcct ccccagagga gcctttgaaa acctcacttt tc #aagacgct    360 tcaactccta ttagaaagtg acttaaataa aattgctctt ctgaccatcg cg #gctctgga    420 tgccaggcta gtgattattt gtctaatctg agtctccagg gctagaactt ca #caccgtgt    480 cttttcacta ccctcccaga ggaggtgaga ggaggtgaat agacacttgt ca #gattcttc    540 tgcctggtgt cactgtgggg ggatgcacac acactggagt ataggcttgc ta #aagagaga    600 ctgcattgct atttcaacta gacacgttta cttttcccgc aaatgattac tg #agcaccta    660 tggtgtgccc ggtaccatgt tagggcaaga gatctacctc atccttgcat tg #taaacctg    720 tagcatccgt ggcaatgttt tacaccactg ccctcccagg gttcctgcaa gc #tgactcct    780 tttattttcc ttgtgcatgc tgatgtgttc aaggtatttc agtgggctac tg #tgagacta    840 tctccccatc aggatctctc aaccttggca ctaggaacat gttgggaaaa gt #aggtctct    900 gttaagggag gaagaggggc tttcctgtgc gtgataggat gcttggcagc at #ccctgtcc    960 agtagcacca catctacctc agttgtgaca accaaaaatg tctgcagaca tt #gtcaaatg   1020 tctcctccct attttacatt taccaaagtg ctctttatgt cctttacagt ta #aggaaact   1080 taggaccagg aagaaacttg ctcaagtcaa acagtgagtt ggcggtagag ct #ttagctac   1140 aatttaagtc ttgtcttccc aacccagtgt ccatcttatg gatccattca gt #ggtttgct   1200 cctaaatgct taataaccaa ttatctggga taaacgaaag ccaaacaaac tt #gctgggag   1260 cgtgggacaa tttccatggt gtaaatactt ccaccatggc tggtttcaag ct #acttccat   1320 ggcgtcactg aatgtgaagt tgggaaggga tctgtacagt tggatctggt ga #gctggtgc   1380 cagccagctc agaatgcccc caagtccatg tgtgctgctg atcacatatt ag #tcttgctc   1440 tcctctggac cttgttctgc caccccccag acacatgccc ttatcactgt ga #tgtgctgt   1500 ggggaaagca gtcacctggt aagagttttt gataacttct ctggataaac at #aaagcagt   1560 tagagtggag ttatccccct cttctgttaa atgaaagttt cttttcacag tc #tttccatt   1620 gaaggaatta gaaaagtcat gggatacctc tggtcttaga gcagaaatga ag #ttccccag   1680 aagggggtcc tggacagaat ggtctcttgg gcagggacta gatgctgtta ct #taccatca   1740 gatgtgtcta ctgtaagatg cttgggaggc atcaaagtgg tgacagtggg gg #gctgttgg   1800 attttaccat tctggaatat aagagaaggg gtgtggctgg tgttttagtc ca #gatagcag   1860 ctgtgaccac tgggagcctc attttccaca cctgcaaaag gagaaatagt ga #tccccttc   1920 ctagtgttgg tgtaaagagt agatgaacct ctgcctgagt ctcagatgcc tg #ctgcccac   1980 ttggttgctt agtggatcgg ctggctgtca acagtgtaag cttatcaagc ct #aaatactt   2040 agttggtctt gactttttca cctcgaccat gactcagtgt catgtgctcc ta #gtcattct   2100 gtctgtggtc caacgttagc ctgggaagca cctgggactg agggaagaac cc #tcagctag   2160 ttattcagcg atccaggttc tcctcctgcc tttgatatca cctcattata ta #accttggg   2220 aaacactttg gtgtgactgg aatgtggata ctcccagggg aagggtagga gc #atggtagg   2280 gcatttggac tttatcatga aggtggtagg aaatacttga agggttttaa gc #agggatga   2340 cacatcatca aatgtgtgtt ttgaaaactt ttttctgcag aggacagggc ca #cagcccaa   2400 gcaggatcaa accagttagg agattgatgt aacagtcccg tcagaaagtg at #gaggtagc   2460 tgggtgcggt ggctcatgcc tgtaatccca gtcaccgtgg gaggctgaga ct #ggcagata   2520 gtttgagacc agcccttggc aacatggtga aaccccttct ctacagaaaa aa #aaaaaaaa   2580 aaaaaaaaaa aaaaaaa              #                   #                   # 2597 <210> SEQ ID NO 43 <211> LENGTH: 3116 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 43 ggtgataatg aaagtggtgg tggtgatggt ggtaatactg gtggtggtga ca #ttggtggt     60 ggtggtgatg gtggtgatac tggtgatggt ggtgatggtg gtggcgctgg tg #accctgac    120 atggggtcca gtagcagtga cagtggatgc aggctcctgg tgactgagga gc #atctcagg    180 ctgrggaggc acctctgatc cccgccactg ctccttaccc cctacagtct ct #cagcaaac    240 ctgctgggcg acagcggact cagatgcctt ctggaatgtc tgccgcagtg cc #catctccg    300 gtttgcttga tctgagtcac aacagcattt ctcaggaaag tgccctgtac ct #gctggaga    360 cactgccctc ctgcccacgt gtccgggagg cctcagtgaa cctgggctct ga #gcagagct    420 tccggattca cttctccaga gaggaccagg ctgggaagac actcaggcta ag #tgagtgca    480 gcttccggcc agagcacgtg tccaggctgg ccaccggctt gagcaagtcc ct #gcagctga    540 cggagctcac gctgacccag tgctgcctgg gccagaagca gctggccatc ct #cctgagct    600 tggtggggcg acccgcaggg ctgttcagcc tcagggtgca ggagccgtgg gc #ggacagag    660 ccagggttct ctccctgtta gaagtctgcg cccaggcctc aggcagtgtc ac #tgaaatca    720 gcatctccga gacccagcag cagctctgtg tccagctgga atttcctcgc ca #ggaagaga    780 atccagaagc tgtggcactc aggttggctc actgtgacct tggagcccac ca #cagccttc    840 ttgycgggca gctgatggag acatgtgcca ggctgcrgca gctcagcttg tc #tcaggtta    900 acctctgtga ggacgatgat gccagttccc tgctgctgca gagcctcctg ct #gtccctct    960 ctgagctgaa gacatttcgg ctgacctcca gctgtgtgag caccgagggc ct #cgcccacc   1020 tggcatctgg tctgggccac tgccaccact tggaggagct ggacttgtct aa #caatcaat   1080 ttgatgagga gggcaccaag gcgctgatga gggcccttga ggggaaatgg at #gctaaaga   1140 ggctggacct cagtcacctt ctgctgaaca gctccacctt ggccttgctt ac #tcacagac   1200 taagccagat gacctgcctg cagagcctca gactgaacag gaacagtatc gg #tgatgtcg   1260 gttgctgcca cctttctgag gctctcaggg ctgccaccag cctagaggag ct #ggacttga   1320 gccacaacca gattggagac gctggtgtcc agcacttagc taccatcctg cc #tgggctgc   1380 cagagctcag gaagatagac ctctcaggga atagcatcag ctcagccggg gg #agtgcagt   1440 tggcagagtc tctcgttctt tgcaggcgcc tggaggagtt gatgcttggc tg #caatgccc   1500 tgggggatcc cacagccctg gggctggctc aggagctgcc ccagcacctg ag #ggtcctac   1560 acctaccatt cagccatctg ggcccaggtg gggccctgag cctggccagg cc #ctggatgg   1620 atccccccat ttggaagaga tcagcttggc ggaaaacaac ctggctggag gg #gtcctgcg   1680 tttctgtatg gagctcccgc tgctcagaca gatagacctg gtttcctgta ag #attgacaa   1740 ccagactgcc aagctcctca cctccagctt cacgagctgc cctgccctgg aa #gtaatctt   1800 gctgtcctgg aatctcctcg gggatgaggc agctgccgag ctggcccagg tg #ctgccgca   1860 gatgggccgg ctgaagagag tggacctgga gaagaatcag atcacagctt tg #ggggcctg   1920 gctcctggct gaaggactgg cccaggggtc tagcatccaa gtcatccgcc tc #tggaataa   1980 ccccattccc tgcgacatgg cccagcacct gaagagccag gagcccaggc tg #gactttgc   2040 cttctttgac aaccagcccc aggccccttg gggtacttga tggccccctc aa #gacctttg   2100 gaatccagcc aagtgatgca cccaaatgat ccacctttcg cccactggga ta #attgactc   2160 aggaaagaag agcctcggca gggcgctctg cactccaccc aggaggaagg at #acgtgtgt   2220 cctgctgcag tcctcaggga gaactttttt gggaaccagg agctgggtct gg #acaaagga   2280 gtaccctgca ttacgtggga tatgtgtgat caattgggga catgcgacac ac #aatgaggg   2340 tgtcatgaca atgcatgaca cgtacggtta tatgtggcag tgtgacccct tg #acatgtgg   2400 cgttacatga aagtcagtgt ggcacgtgtt ctgtggcatg ggtgctggca tc #ccaagtag   2460 caggatacat gattgttggt ctatatatga cacatgacaa atgtccatgt ca #caggactc   2520 atggctggcc agatgacctc aggctggccc aagatctaat ttattaattt tt #aaagcaaa   2580 tacatattta tagattgtgt gtatggagca gctaagtcag gaaaagtctt cc #gcccgagc   2640 tgggagggga gagtgtccat gcactgacca gtccaggggc tcaagggcca gg #gctctgga   2700 acaagccagg gactcagcca ttaagtcccc tcctgcctca atcctcagcc ta #cccatcta   2760 taaacttgat gactcctccc ttacttacat actagcttcc aaggacaggt gg #aggtaggg   2820 ccagcctggc gggagtggag aagcccagtc tgtcctatgt aagggacaaa gc #caggtcta   2880 atggtactgg gtagggggca ctgccaagac aataagctag gctactgggt cc #agctacta   2940 ctttggtggg attcaggtga gtctccatgc acttcacatg ttacccagtg tt #cttgttac   3000 ttccaaggag aaccaagaat ggctctgtca cactcgaagc caggcttgat ca #ataaacac   3060 aatggtattc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa ct #cgag       3116 <210> SEQ ID NO 44 <211> LENGTH: 3460 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 44 acgcgtccga tgaacttgac cggctaaatg ccccactttc tcagatggct tc #taacgact     60 ttcaggatta gggccagctg tgggtctact ccttgttgga gcccatctca cc #tgggatgc    120 ctgcagccag ccctccctcg tgatttgtct caccttgagt aggagacatg ct #tctcccct    180 aaccttttcc tttctgccat aattaacata tgtccttttc agtaagtcca tg #cctctggc    240 aggggatgaa gaagtactca ctggtaatta gctaccatct ttgcagcagc cc #tggtaact    300 tgaaaaattt gggtctggtg ctgttcatga gtctttgtgt aactgcaaaa gc #aggaaagg    360 aagtcaagac tcctgttgcc tcgtgcttag caaagcagtc cttatccttt at #actctgtt    420 cttgggtttt gtttttgtct tgttttatac caggcaaatt gcttagtagc aa #agggacca    480 aactgaaaag gtgacaatct ctaacttcta aaagcagaca ccaatcggat gc #tcattaga    540 ggttaatgaa gatgccattc ttggtggcct ctgcacccaa attgcatctg ga #aagaacta    600 gggtctcatt cagaatgtcc aaaaggaaat tcttaagagc ttaaattcag at #ttgtgtct    660 cattaatgca gtgaacaatt caaaaccaca cagattcctt ggcaggaagg at #aatggaat    720 aacagtgttg atgagacctt tttagcttca aggtttcgga gtctaaacaa at #ggatgatt    780 catttggaat gaaactcaca atgcaagtag aaggacctct ccaaatcagg cc #agttgggt    840 tatcctggct tggaatctgg tgtgaaacca taggtcttaa cactctggag ca #gcacattg    900 ctgtggatat gtccaggaga ccttagatat ggcttaaagg ctttcaagat ga #ggacagaa    960 attgcttaca attgctcagt ttctcaacag aaagactcat aagagtgcca gc #atggggta   1020 catggagtga agctgggtgg gaagcatcat ctgcacagtc cctgtcctag tg #caggactt   1080 ttctctgtat gttttcatac catgggattt ttggatatca gtgtattttg gt #tcttgaaa   1140 tagcctaata gctgctcaca cattgggtag gaatattata ccaatgtcat cc #ccaaagga   1200 agggtgagct gaatggaaat taagcccagt cattttattt gatctattag ct #ctgttatc   1260 agtgcatgat cacccagatc accctcctca gcccacacag tgctgaacca tc #ttccctcc   1320 tgttctccat ggctattaat agtatagcta aatttagagt gcagagccag at #ataagtat   1380 tttggaatta tctcccagtt tgtggtagaa gctgactgga atacaggttg ag #tatctctt   1440 atccaaaatg ctagggacca gaaaggtttc agattttttc agattttgga at #acttaaca   1500 gttgagcacc ccaaatctga aaggcttctg aacgtcatgt cagcactcaa aa #aagtggat   1560 tttggagcac ttcaaatttc ggatttttgg atttgggatg ctcatcctgt gt #aggagagg   1620 ctactcgatt ccatttaatg actgtcctag tcataatcat ccaaagataa aa #gccaggta   1680 gatgttgaaa gctctttcca gggctgaaaa agtgttctta cgttctctgc at #gtgactag   1740 catcactgtg gaaattaatg ctctgttctt cactagaatg tagtaagtgg tt #aaactgag   1800 ctatccccca cctgatgact attggcatcc atttgcaagg ccaatggcct gg #attaaggg   1860 ttaggattat ttgtagctag aaggtaattt tatttctgtg aaactaattg gc #tcatattt   1920 gaggttaggt gtggccttga ccttaccagt acatttatac ccactaccag tt #gactagcc   1980 cagataattg ttaaatggtg cttcttttct gcttctcagt agacttccat gc #cattacaa   2040 aggaaatttg aattacctag tgtttgtata ttccatgata actatgtata ac #ttctgtta   2100 cacagcttat gtattgttaa catttaagtg taaaccatgc cacagctaac ac #ttaaaaat   2160 gaaaactaat tagttcttgc ttagggaaaa tgccaggtat gaagtatggc at #atacttga   2220 cactgtcctg tgtaaccctt tactttgctc aggctttcaa gattgagtct tt #tttccccc   2280 aaattaggtt aacatgcatt tgaccccaac ctgtggggtt tgagtaagct gg #aaatctgt   2340 gacggtaggc tttctagtgt cacgaggtgg tggtgactga aggaaaagct gg #gatcacag   2400 gttccttctg atggagagga aggtttattt ctatgcccct cccaccaccc tc #cacctaga   2460 gctcacccaa gcctgctcca gtcccagggg caggccattc tgcaaaagca gg #acctcaca   2520 gaaacaaggg ctgggttgag gtcaccccct tcagagttgg ttcctggcca ga #tgggtaag   2580 aggcatttgt aattttaaaa atgtgaaact tgggtttggt gttttcttct aa #gtgcctaa   2640 ataagcaagc caggctgttg atattttagc cagagaaatc ggcaagccaa ga #ttaacccg   2700 aatctgaagt ttagaatctt gagtttgcat ctgcatcata tcatgctgtt tt #gatgagga   2760 aacatttgcc actgaggagt tggagggagg gcaagacgac agtgttaagt ca #gatcattt   2820 aatggtttcc cctaagccct ggaaaaatat ttgaaagaat ggcagcaaaa ag #gttaagaa   2880 agcaagccag atttactgca caatatgcag tacccagtac tactttaaat cc #caagagaa   2940 cagtgtgatg tctaatatat acaggtctat gaaaatactg tggaataagc cc #aggaaggt   3000 tagatgtgtt tgcaaataag ttgcccaaag ggtccccctc taagtaaaac aa #atattcag   3060 accacaggct ttaatgtaaa ctgtcaaaaa gtgggatgtg gaggattttt gt #taagtgtc   3120 aatcgaagtt aaaaagcaag ggtttttggc caggcgtggt gctcacgcct gt #aatcccag   3180 cactttggga ggccgaggcc ggcaaatcac ctaaggtcag gagttcgaga cc #agcctggc   3240 caacatggtg aaaccccgtc tctactaaaa atacaaaaaa attagcccgg tg #tggtggca   3300 agtgcctgta gtcccagcta cttgggaggc tgaggcagga gaactgcttg aa #cccgggag   3360 gtagaggttg cagtgagcca cgatcatgcc actgcactcc agcctgggca ac #agagcaag   3420 actccatctc aaaaaaaaaa aaaaaaaggg cggccgctct      #                   #  3460 <210> SEQ ID NO 45 <211> LENGTH: 2622 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45 aggttccctc ctttggatat ttggcagttg tactttatgc agttcagcca ag #aactaaac     60 acaggccttc aggtgtggtc agagtacagt gggccagagg tcctttcttt tc #ttcatgtg    120 gtcatacacc tctgttaatt caacttaagt ttatattact tttgggggtg gg #agtgggga    180 agaggatcac attgttgact tgctggaagc attatgtcaa ctaaaatcct tc #agttctta    240 ttttcatcat gctgttgggt tccccctatg ttgtttcttt ttaaaaacac ca #aatgcaga    300 acttcccttt tatactgctt ctatttcatc ttgttgactt gtagcctatc ag #agtatgat    360 tctttattgt catctaaggt attctgatat aatcactgca tgtctaaatt ct #agttattg    420 actaaatgct acttgagacc attctcttgg ttgtcacatt attttacagt tg #aagaaact    480 gaaccttaga gaggttaagt accttgttaa aggccacctg gctggaacat aa #catcctgg    540 tcattttaac tactaatgct gtcatgaaag ggagttagcc agggcaggtg tg #aatagagc    600 attctagcat aagcagccaa caattagagc aactgtgtat ttttagtgtg tg #gatagcaa    660 atctttgctg tcttggaagt tactatctta gaagcatggt attaagctct ct #attcaatg    720 tggtgttcac atcagtagca ttggtttcat ttgggagctt tttagaaatg ca #gagctcag    780 gccccactcc aggcctaagg attcaatctg cattttaaga agtgacccat at #acacatta    840 aaggtttaag aagcattgct gtagtatcct attttccaca ctttagtcat tt #gcatatca    900 cttgtgcaac tttggcttta tctgcatact tcctgaaggg ggcctgcccc tc #cacacctg    960 tgggtatttc tcgtcaggtg gagatgaaag aatgagaaaa gaaataagac ac #aaagtata   1020 gagaaagaac agtgggccca ggggaccggc acactcagca tgcgaggacc tg #caccagtg   1080 ccggtctctg agttccttca gtatttattg atcattattt ttactgtctt gg #cgaggcga   1140 gtgtagcagg gcaacaggtg gcgagaaggt cagcagggaa acgtgagcaa ag #gaatctgt   1200 atcatgaata agttcaagga aaggtactgt gcctggatgt gcacataggc ta #gatttatg   1260 tttcacttta cacaaatatc taactagcag agagcaacaa agcagtattg ct #gccagcat   1320 atctcgcctc cagccacagg gcggttttct cctatctcag aatagaacga at #gggaatgg   1380 tcgactttac actaagacat tccattccca gggacgagca ggagacagaa gc #cttcatct   1440 tatctcaact gcaaagaggc ctccctcttt cactactcct cctcagcaca ga #ccctttac   1500 atgtgtcggg ctgggggatg taaggtcttt cctttcccac gaggccatat ct #caggctgt   1560 ctcagttggg ggaaacctgg acaataccca ggcttttctg ggcaggggtc cc #tgcggcct   1620 tccgcagtgc attgtgtctc tggttaatcg agaatggaga atggcgatgg ct #tttaccaa   1680 gcatactgcc tgcaaacata ttgttaccaa ggcacatcct gcacagccct aa #atccatta   1740 aaccttgatt caatatagca catgtttctg ggggcacaga gttggggcta aa #tttacaga   1800 ttaacagcat ctcaaagcag aacaattttt cttagtacag atcaaaatgg ag #tttcttat   1860 gtcttccttt tctacgtaga cacaggaaca atctgatctc ttttccccac ac #ttcctata   1920 tgttaatgta ttttaattgt ctgttagctt ttatgtcttt ccaacttgaa at #aatttcac   1980 ttacaaaata gctggaaaaa tagtaaagat aattcctgta ttttcttcac cc #acgtattc   2040 ccaaatgtgt aacacctaca atcagcaaaa atcagcacat taatattatt ac #ctaatcta   2100 cagacctcat tcacattttg ccagttgttc tgctggtgtg ctgtatgtag tg #caggatcc   2160 agtctaggcc cgtatgttgc attttgctgt catgtcttct tagtctcctt tc #atctcagt   2220 ggtcttttct tttccatgac tatgacagaa atgatttgta tccttcatat at #ctgcagtc   2280 acatgaagtc tatttgtccc actactggtg acgtaaattt tggtaacttg gt #tgagggtg   2340 gtaccgtcta ggtttctcca tattaagttg ctatttttcc ttgtgtattt aa #taagtatc   2400 ttatagggaa atcctttgag actatgtaaa tatcatatta catgtatttt gc #ctatttta   2460 gcatcttttg aagataattg cctgaaacat tttttactgt gatgattttc ta #attctatc   2520 attctgcatt tattagtttg gaattcgatt gtaaggaagt atattccctt gt #ttcctatt   2580 attttattaa agaaatttac atccaaaaaa aaaaaaaaaa aa     #                   #2622 <210> SEQ ID NO 46 <211> LENGTH: 1984 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (106) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 46 agttgttgct atgaatgatt ttaagccttt ttttttaagc ttggcaaaca tc #ccagctaa     60 tcaaaatagt catattcctg agaagtagga aactaaaact tctttncata ta #attgttag    120 aaggtttgtt tcccaaacta ccatagttac aaaggtgaat agccaaattt ta #ggaacaga    180 atcaaaagaa taaaaatctg tgaagagatc ctactactct tccttctatg tt #ttggtttt    240 ggtttctatt gtccctatca tttcagcaag tggaacagca gcaagttttt ca #gtgcatat    300 gctgcacaag aacaaaatat aaatctgtat ggcaccaaaa atcaaagtga aa #accaaacc    360 aaaaacccaa acaccctatg taactatcgg aggcatatac gtggtataaa tg #actgtagc    420 tgtgatacac acatggctac ttgtcacatc actttccata attatttact gc #aaaatgat    480 tgagaggctt ttggtgcagg cagccgttaa cctcctgctt cctttgttac ct #ctggatta    540 ctttgcagta aattgcaggt cttttaagag atttaagctt cagttttctc aa #accaaacc    600 aattatcctg tcttatctga agatgcaggg ttgtgggcaa aagaggctgg tt #ataataat    660 gccctcatat tgagtggtct gtaaacggct gcacacttca ggcactgtag tt #gctgaaga    720 tgctttgtta aatgtgacct tgactggctt tacaggggtg tagaatgtaa tc #tacacaag    780 gtgactttgc atctatcttg ctcttgaggt ggatgaaatt gagaagctgg ag #tgtgtaag    840 ccatgcacat aagtattctt cactgtaaat tttgttttca tttttaaccc aa #ttatggta    900 ctttatccaa tgcacaactg atctctcagt agatattcat ttgaaaatag tg #tggccttg    960 atcagtgaga aagggaagga gaaaagtgac ttttttgctt atgtagaaat ga #ctcatttg   1020 ctgagagttt gtctttctgc agcactcttg gtataatgtt agtgatcggt ct #cctttttg   1080 attggggaaa gttaatgttt ttgaccctgg agttaattca gttgagttat ct #tatatttt   1140 taggaagtat cagaattgct ctgatgaata acaaagttga ctgttttgat gt #ccaatctc   1200 aggttttaga atatagtggt gtaaagtccc actattttta attcttaaaa ca #actttaat   1260 ttcgtacacc ctaaaagtca catgcataag gcctgttcag agagcagagc ct #ccatcttt   1320 ttgctccttt tctactttgt acttcacttg raaaaatatc aagtgacttt ac #atkgtata   1380 tttccattgt aaccctgaca tttctcaaag ataaagcact ttttgatcat ga #aatacatg   1440 aaatctttgt gtgatgtgga tcatagtttc tcaggctccc ttagataatt gc #ttatgaat   1500 attgttctaa ctctgtgtaa gaagagtaga aatctttgct aatgttagaa gg #tttgtatt   1560 attgatccag aatgcatttt gctagtttcc aatggatggg agagtaaata at #gctgcatt   1620 cacaatttaa taagttactt tcccttgagc cttaaggtaa ctttttcttt tc #tgtcaact   1680 acagcactga agttatagta agtgaatgag attatcagtt ttcagggttg gt #tttagagt   1740 actgtaaatc aattagctgt cttcctaaag agttacaact cccattcagt at #actggata   1800 atgggtgtgt gggtggggct ggggagggcg ggagatagtt tgtagaaaag aa #aaaagaaa   1860 aaaaaaaaaa acacatttac cttaagaaaa tacagacaaa aaaaaaaaaa aa #gggcggcc   1920 gctctagagg atccaagctt acgtacgcgt gcatgcgacg tcatagctct tc #tatagggt   1980 cacc                  #                   #                   #           1984 <210> SEQ ID NO 47 <211> LENGTH: 1987 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (442) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (444) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (473) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (493) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1011) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1025) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1111) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1119) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1169) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1234) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 47 ggcacgagac cggcgctagt cttgcgtcgc acagcaggat tgcgctgctt tc #tgatgacc     60 ttcgacatgg atggcgatgg taatgaacgc ttttcccgta acgccccacc cg #caaccgac    120 ccaggccgga atgaccctga tcgaggtcct ggtatctgtg ctgatcctcg ct #gtcggcct    180 gctaagggcg gcagtcattc agctcaatgc attgaaatac accgacagtt cc #aggatgac    240 cagtcaggcc agtttcattg cctacgacat gctcgaccgg atccgcgcca at #tcgggtgc    300 tgattactcc tggggccagg gtgaacgcgc gccctccacc acctcggtcg cg #agtgtgcg    360 tgatctggac ctgcacgact ttgaagcgaa tatcgtcggg ttcgccgggg aa #agcgccaa    420 ggggtccgtt gcggtcaatc ancnagaagt gaccatcagc atcagttggg ac #nactcccg    480 tggagcgaat gcncaaggca cccgggaaac attcaccctg accagccggg tt #gcagtcga    540 tccgagggtg ttgccatgag aggtccagtg tgcggtttca gcctggtgga ga #tgctgctg    600 gcattggccc tcggcctgat gttgatcctg ggggtgaccc aaattgcact ca #gctcccga    660 accacttatg ccagccagag tgcggcttcg ctgttgcagg atgatgcacg gt #tcgccctt    720 ggcaagctga ttcaggaaat acgccaggcg ggcatgtttg gctgcttgtc cg #ctgcatca    780 atcagcaacg ctcccgcagg ttttgatcgt cccattggat ggagtaccac cg #gcagttcc    840 cggtccctga cgctggtgac cgccgacgtc ggggagggtg gcagcaagcc gg #actggacg    900 gtgctttccg attgcaccgg ctctgcccac gcctatgttg gaagcccgcc gg #cagcgaac    960 gcccgggcaa atccacttcc cacttgcgca aagctgacct aacacctttg na #gggcgggc   1020 aagcnggaag ttaagtaacg ctggcggccc ccgagcaaag cggtggttgg tg #gataacgt   1080 ggggcattcg atatcagttt tcggcgtggc ngacaagcnt ggctcaacgg tt #gtcagccg   1140 atatgacccc acccccggcg atgagtcgnt catccgcagc gtgcggattc tg #ctgacact   1200 tcaggatcca aatgggttgg tgaaagacca ggcntacagc gtggtcgcgg ca #ctacgtaa   1260 tcgcctggag tagcgtgccc atgggttatt acctctcccg ttcgaggcag gc #aggcatgg   1320 ttttgctgat cagcctggta ttcctgctgc tgttggcact cctcggagtg tc #ttcgatgc   1380 agggagcaat ctcgcaagaa aaaattaccg gcagccttcg gcagcgcaac ca #gtcgtttc   1440 agcaggccga aagcggcctc aggcttggcg agtctttggt gcaggcgtca gg #tttcgccc   1500 tgcgcccttg ccactcgacg gctgcgtgcg cgccacctgc cgaatcggtt tc #ggtagtgg   1560 ggccggggac gaaccccgta tcgactgtga cctggatagg gatgaaagat gg #cgtctacg   1620 gtattcaaaa cctggggccg ggaacgggtt tggtcaactc ccggcagagg cc #caggccac   1680 ggtctatcgc gtgacatcag tgggcgtcag tgggcactcg cgttcggtcc tg #gagtctgt   1740 gtatgcccgt gtgggcagcg ggcccggcga gcgtttccga cgaatcatgt gg #cgacaact   1800 tcaataggtg agcagcacga tgggcaagga ttgcacaggc ttcaccctga tc #gaattact   1860 gatcgccgtg gcctcgtgcc gaattcggca cgagattaat tcccctaaaa at #ctttgaaa   1920 tagggcccgt atttacccta tagcaccccc tctaccccct ctagagccaa aa #aaaaaaaa   1980 aaaaaaa                  #                   #                   #        1987 <210> SEQ ID NO 48 <211> LENGTH: 2113 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 48 ggggaatatg gatggctttc ttctttgccc tctttgttat tttctttgtt at #tgttgttc     60 agatggagtc tcactccggc ctgggcaaga agagcaaaat tctgtctggg gg #gcaggggg    120 aagaggtata ttttttggat taaaatggaa tgctttcctc aaactgttaa aa #ttaggtag    180 tgttacaagt ttaaagaata ggtttagcca gtgtgattat tgcagtgtaa ta #aaaaggat    240 cccaggtggc acacatgcta gtgtggtgcc cagatgctca actgaagtga ag #gataacat    300 gcgtttcaac attttctata tactaggcag tttttcttaa gtcttggttt at #tgtgatat    360 aatacagtaa aatttactct ttatacgtat gattccatta atattaacaa at #ttaaacag    420 tcttacagct aaatacaatc aagatacaga acatttccat cactacagga ag #ttgttttt    480 tgctaattta actaaacttc ttaccatgct caacccccca cccccaccct ga #gaaccact    540 gctttgtttt tctgatcata tagtttggcc ttttcaaawa tgtcatacag tt #ggaatcat    600 atagtatgca tgaatcatgt agctgaagtt ttcatttcac ttaggtaaac ac #ctaggaat    660 aggagtgctg ggttatactc taagtgttaa acttcataag aagctgctaa ac #tgttttcc    720 aaagcatctg taccattttc ctttcccacc agcaataaag cattcattag tc #tacgtact    780 caccagtgct agtgtggtca gaatgtatag ttttaattat acacattata at #agatgtag    840 agtggtatct catcgtgatt ttgccttttc ctaatgaata tctttcctta ca #tatttgtc    900 attagtgtgt cttctttagt aaatggtcct attgttttgc ccatttttaa aa #gttgagtt    960 ttcatattgk tcaatttcga gagttcttca tatattctgg acacaagtcc tt #tgtcagac   1020 atgtgatttg caaatatttt cccccagagt ttttcctgtc ttgtcagtcc ct #taatagtg   1080 tttttaggaa ggagaacagc tgcttttaat tttgataaag cttgcaattg tt #tttctttt   1140 atggatcatg cttttggtgt agtatctgag aactctttct taaaccagtc ac #acatgtct   1200 tctgttttct ttgaagcttt acaggttgag gtacattttg gtccatgatc ac #ttttgagc   1260 tactttttat atataggtag tataagatat ggtttgaggt ttttgtttgg gg #tttttttt   1320 cttcctgcat atgaatgttc agttgttcca gcaccatttg ttgaaaaaac ac #tatccttt   1380 ctctagtaat ttgctttttt acttttgtca gttgactatt tgtgggtctg tt #tttaaact   1440 ccattttata ttatgtctac attatgaact ttatagttag tcttgaaatt mg #gtaatgtg   1500 ggtcttctca cttgtgcttt tcaaaattgt tttggctctt ctaattcttt ca #ctttttcc   1560 atataaattt tagaaamagc ttattgattt ctaccacttt cccccaaaaa gc #cacttggg   1620 aatttgacta agtttacatc gaatctaata aatgattttg gagagaagtg gt #atcttagc   1680 aatacagtct tttctgataa cacttcctgt tttagttctt tttctcattt aa #taattttc   1740 caacattaaa accttggaca aatttagatg tgtatctgaa taattccagt tt #tgttgcca   1800 tttaaaatgg tacataatcc ttattttata gatgagaaaa ttaagtaact tg #cccaagtc   1860 acacagttac taaatgacaa agctagattg aaatctatga gcttataaac ta #atgtcctg   1920 ttttgagcta cagtactagt tttattaaat tgttgtggtt aaactgaggt gg #gaggatag   1980 tttgagcacg gaagattgag gctgcagtga tctttgatcc tgccattgta ct #ccagcctg   2040 gcaacagagt gagaccctgt ctccggaaaa aaaaaaaaaa caccatggaa gc #aagcaaaa   2100 aaaaaaaaaa aag               #                   #                   #    2113 <210> SEQ ID NO 49 <211> LENGTH: 3465 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 49 agaaaatgaa agcaccaaag agccttcttt gctacaatat ctttgtgtgc ag #tctcctgc     60 aggattaaat ggtttcaatg tacttttatc tggcagtcaa acccccccta ct #gtgggccc    120 gtcctcaggt cagctgccgt ctttcagtgt cccttgcatg gtcttaccat ct #ccacctct    180 gggccctttt cctgttctct attctcctgc aatgccgggc ccggtttctt ct #actcttgg    240 tgctctccca aacacaggac ctgtgaattt cagcttgcct ggccttggat ca #atagccca    300 gcttctcgtc ggccccacag ctgtggttaa tccaaagtcg tccacactcc ct #tctgcaga    360 ccctcagctt cagagtcagc cctcactaaa cctaagtcca gtgatgtcaa gg #tcacacag    420 tgtcgtccaa caacctgagt cccccgttta cgtgggacat ccagtctcag ta #gtaaaatt    480 acatcagtca ccagttccag tgacccccaa gagcatccaa cgcacacatc gt #gagacgtt    540 tttcaagaca cccggcagcc ttggagaccc tgtcctgaag agaagagaaa gg #aacaatca    600 cgaaacacca gctcggccca gaggagacta gaaatcccca gcggcggcgc tg #actaacct    660 gccgctttgc caggtggggg tgggatcaaa cgccctgaga gtcccggatg tc #cgaggcgg    720 gatgcaaacc atcccgtcct gagcacgggt ccttcctctc tctttcatcc ac #acttctgt    780 taacttccca ccaccatcaa tcatctgatt tcctgaaagt aattaattgt gc #atttaata    840 ccagttagag ttccgactct gcatggtgtc acagtgaaag cgccgactga ct #tatggttt    900 tgattcaaga atcgtcttat tgctggaagt agatctgaat aggataccgg ag #ccttgttt    960 ttctaaaggg gggcgctgtc tagcacttaa ctagggtaag cattcttaac at #gtatttcc   1020 acttgccctg agtaaatctg tggtgagaga agcttccttt ctgcagttta aa #aaagctac   1080 tgcttcctta ggcttcatca ggaagccatc ttcagttgtg aatcctatgg tg #ttatttat   1140 tttgttcctg aaatgggatt tagtgcaaaa agtttacaac tacagtcttt aa #cacatttt   1200 tttcagggta tgacgacttg aatgtttata cttttattct ataatttgcc ct #gcacttat   1260 tttacaacct agtaataatg tggataaatg tatctacatg acacatgtca ag #accaaaat   1320 aactgtgaat gacacacctt gctgtaaatg aactgtgcta accctgactg tg #ggcttgag   1380 aacaaagatg aactctagaa ctctagcagc ctaactgctg cttctcaaat aa #ctgtgtga   1440 acagtgagat attactgttt gtttctaaaa atcctactgt gcccagtttc ct #tcactaca   1500 tgccctgcat tttttattta aatatttagc tgtagcgcca tcagatatgg at #gccttcta   1560 acaattgctg tttgtaaaat aaatcaggat ggtagaaagt gattttatgg aa #aattggaa   1620 cctggatgag accttttcgt tgaattctga agagtaatga tgcgaaaatt ga #tacagggc   1680 aagagatgat tcttttgttt ttcttctact tcatgtccag aagagtaaga gg #gaaaatgg   1740 acatatgttt catatccaag ggtattcaaa ctgtagttag ttggtacctc tg #aaaaatga   1800 gaatggtgag cgcacgggtt ggttgttcta gcatgaatac aattctggaa ac #tgttatgc   1860 aatttccctt ttttaaccca cattacttta ggggtgcatt aagtcgccaa ac #tatactag   1920 ttctttgtat tcctagactt gctgatattt acctctctct tgtctcttca ga #gtaaatgg   1980 ttcccttctt tccttcctac tttccttcat tctctcttcc ttccctcctt cc #tacttctt   2040 ttcttccttc ctcttcctct cttaaaacta tcttagatgt agaatcctgg tg #tagggttt   2100 tattttattt ttattttttg acccaataaa atgttatatg aaagaatgaa aa #tattaatt   2160 taagagactc tgggagtctg aataaagtag ctttatatta actacaggat aa #tattagcc   2220 ttattacccc cacaagattt tttaaaactt gaggtaggta gctacattaa at #aaatttgc   2280 tacttatata aaaattttta tcaacactaa acttttaaag tttacaagtt tt #ttttttct   2340 tttttacagt cttctataga gttaggttaa aaatgtggtt ctaaccatca ac #aattgcat   2400 ggttaaatga ccctgaacta aaactgatgg gttccctatc aaaacaaata aa #aatatacc   2460 tttttcaggt ttcaatctgt gcagggtata tgcatgttaa ttctaccatg ct #taagaact   2520 tccacaaaat atttcatgga gaggtctgca tttagacgga aacagaaatt gc #ttttcccc   2580 tcactgttcc tgaatgctct atacttgttt taacattttt gctatctttt tt #tattattc   2640 tgatcatgat atgaccattt aacctcagaa ttcataattc ctgaggggtg tt #aagaagca   2700 gtcccattgg tgaggatatt atgacttggt gaccattctt aggagtagaa aa #ccaaggac   2760 aattgcttct gtattcagta tccacttctt aatgtggctt tatatgtaaa aa #taataatg   2820 cagtggttgt ttctgtcagg aaaataaatc ttacagaaca actggtggaa tt #gaagctgc   2880 tgcgctagac ttggatattt tgggtagtga agaagcaatg gcaatcttga gt #ctattatt   2940 gtataattta gtaaaagaaa aaaataatcg ttggtggtcc tactaagaga at #gcagcttt   3000 tttgagttgt cacagaggct gtgtgtgccc tacactgacc agggtttgta aa #accctttc   3060 attctggtac aagagtcggg ggtataactt ttatacttga atctacctac ca #agtttaca   3120 tttctcaatt cctttttgta aggtgctatt tctgtattta aataactttc tt #ttaacgta   3180 aagctgcttt ctgcttatct tattgcactg ctagttgtat gtaggtatta at #tttattgc   3240 tgcttactgc ttttgttttc ttattattta gctctgctct ttttcctaat gg #ctatatta   3300 tctatagcta tttacttgta actgtactac atgtaaactg attttttgtt ct #gatttttt   3360 ttctaatatt tttaggaaaa tattaagctt tataaaatag caataaaaaa ta #attcattt   3420 aaaaaaaaaa aaaaaaaact cgtagggggg gcccgtaccc aattc    #                3465 <210> SEQ ID NO 50 <211> LENGTH: 1237 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 50 atgggccagg aatggggtcc ccgggcatgg tgctgggcct cctggtgcag at #ctgggccc     60 tgcaagaagc ctcaagcctg agcgtgcagc aggggcccaa cttgctgcag gt #gaggcagg    120 gcagtcaggc gaccctggtc tgccaggtgg accaggccac agcctgggaa cg #gctccgtg    180 ttaagtggac aaaggatggg gccatcctgt gtcaaccgta catcaccaac gg #cagcctca    240 gcctgggggt ctgcgggccc cagggacggc tctcctggca ggcacccagc ca #tctcaccc    300 tgcagctgga ccctgtgagc ctcaaccaca gcggggcgta cgtgtgctgg gc #ggccgtag    360 agattcctga gttggaggag gctgagggca acataacaag gctctttgtg ga #cccagatg    420 accccacaca gaacagaaac cggatcgcaa gcttcccagg attcctcttc gt #gctgctgg    480 gggtgggaag catgggtgtg gctgcgatcg tgtggggtgc ctggttctgg gg #ccgccgca    540 gctgccagca aagggactca ggtaacagcc caggaaatgc attctacagc aa #cgtcctat    600 accggccccg gggggcccca aagaagagtg aggactgctc tggagagggg aa #ggaccaga    660 ggggccagag catttattca acctccttcc cgcaaccggc cccccgccag cc #gcacctgg    720 cgtcaagacc ctgccccagc ccgagaccct gccccagccc caggcccggc ca #ccccgtct    780 ctatggtcag ggtctctcct agaccaagcc ccacccagca gccgaggcca aa #agggttcc    840 ccaaagtggg agaggagtga gagatcccag gagacctcaa caggacccca cc #cataggta    900 cacacaaaaa aggggggatc gaggccagac acggtggctc acgcctgtaa tc #ccagcagt    960 ttgggaagcc gaggcgggtg gaacacttga ggtcaggggt ttgagaccag cc #tggcttga   1020 acctgggagg cggaggttgc agtgagccga gattgcgcca ctgcactcca gc #ctgggcga   1080 cagagtgaga ctccgtctca aaaaaaacaa aaagcaggag gattgggagc ct #gtcagccc   1140 catcctgaga ccccgtcctc atttctgtaa tgatggatct cgctcccact tt #cccccaag   1200 aacctaataa aggcttgtga agaaaaaaaa aaaaaaa       #                   #    1237 <210> SEQ ID NO 51 <211> LENGTH: 1397 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1383) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1396) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 51 ggtccctagg agttgagcag gaacaggcat ctgtggttta cggcgacctg gc #tctccgcr     60 ggccacgtgg gtggtgaggg cacacgagtg ggaagcggca ccgacgtgtt tc #tccccgac    120 cgtggctttg ccaaagactt ttaatagcat tttttaagtg caaaacgtct ag #gtaaaaat    180 ctttatcatc agtgaccaaa ttagaatgta tttaatatag taggtggttt aa #gaactgtt    240 ttaacgtaag acaaactgat agcaacattc tgttgtttta aaggaagtgg gt #ccgtgaca    300 ttctgcagct agtccactac tccaaggtaa ctatcgactt ggtttcagtg aa #tctatttt    360 gtttttaact acagtgattt attagctcag tatctagaaa ttacgtatat tt #tgtgctac    420 tgtcatcgat gtgtaaactc tgtttttatt tgtatttatg cacttggttc cc #atttggag    480 cctctggtct tttctgggat aagtggtgtc tgccgagaca tctcccggtt gt #cagtggtc    540 aggagcagct gagctctagt ctgccagctg ctctgctctt tctgggaagg ag #gtggcgcc    600 cgcccctcag ggtgtctcca gggctcagct tccggggtgg tagagctggg ga #gccccagg    660 ggtgggggga cagctgggag atggaggtgg cacctgctcc cctagatcag ta #ctggctct    720 gaggacaggt gagcagtggg aagaccaaag aatggctggc agcgctgcca rg #gttggaaa    780 tgggggcaag atcctggggc tgtgtgccct ggggcctccc tcacctgtct tg #gtggccat    840 ggcctcaggg atggctccta ggtggctgag gcacagcagt ggctggaagg tg #ccccgtgg    900 aggctgaggt ggaggcgcgc ccagcagctc ccccctgtgg ccatggcggg ca #cgggscgt    960 aggagctggc tggcggccgg ctctgcatgt tcttgttgcc tgtcgtctgt aa #ctctagtg   1020 ttcgacattc gccgtgatac agtggtgtca cgacgtgtgt aactgtggtc ag #cagacctt   1080 gttccgcgtg gacgcctcaa gtggattaat ttctggaagc ctcaatctgt at #gtttgagt   1140 atttacatga gaatgttatt tgaatggaat tttcttaacc cagaaggtag ta #tttataat   1200 catttacttg tagcgaactg tttaaagtta acacttgttt aaattttttt ac #actatagc   1260 atttatgcaa tggtttacag aattcatgga gttattttta tcagtatggg aa #ttaattaa   1320 aaccttgaat cttaaaaaaa aaaaaaaaag ggcggccgct ctagaggatc ca #agcttacg   1380 tangcgtgca tgcgana              #                   #                   # 1397 <210> SEQ ID NO 52 <211> LENGTH: 2271 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 52 cggcacgagc ggcacgagta tcggagggct ttggaccatg aggaggaggc cc #tgtcatcg     60 ggcagtgtgc aagaggcaga agccatgtta gatgagcctc aggaacaagc gg #agggctcc    120 ctgactgtgt acgtgatatc tgaacactcc tcacttcttc cccaggacat ga #tgagctac    180 attgggccca agaggacagc agtggtgcgg gggataatgc accgggaggc ct #ttaacatc    240 attggccgcc gcatagtcca ggtggcccag gccatgtctt tgactgagga tg #tgcttgct    300 gctgctctgg ctgaccacct tccagaggac aagtggagcg ctgagaagag gc #ggcctctc    360 aagtccagct tgggctatga gatcaccttc agtttactca acccagaccc ca #agtcccat    420 gatgtctact gggacattga gggggctgtc cggcgctatg tgcaaccttt cc #tgaatgcc    480 ctcggtgccg ctggcaactt ctctgtggac tctcagattc tttactatgc aa #tgttgggg    540 gtgaatcccc gctttgactc agcttcctcc agctactatt tggacatgca ca #gcctcccc    600 catgtcatca acccagtgga gtcccggctg ggatccagtg ctgcctcctt gt #accctgtg    660 ctcaactttc tactctacgt gcctgagctt gcacactcac cgctgtacat tc #aggacaag    720 gatggcgctc cagtggccac caatgccttc catagtcccc gctggggtgg ca #ttatggta    780 tataatgttg actccaaaac ctataatgcc tcagtgctgc cagtgagagt cg #aggtggac    840 atggtgcgag tgatggaggt gttcctggca cagttgcggt tgctctttgg ga #ttgctcag    900 ccccagctgc ctccaaaatg cctgctttca gggcctacga gtgaagggct aa #tgacctgg    960 gagctagacc ggctgctctg ggctcggtca gtggagaacc tggccacagc ca #ccaccacc   1020 cttacctccc tggcgcagct tctgggcaag atcagcaaca ttgtcattaa gg #acgacgtg   1080 gcatctgagg tgtacaaggc tgtagctgcc gtccagaagt cggcagaaga gt #tggcgtct   1140 gggcacctgg catctgcctt tgtcgccagc caggaagctg tgacatcctc tg #agcttgcc   1200 ttctttgacc cgtcactcct ccacctcctt tatttccctg atgaccagaa gt #ttgccatc   1260 tacatcccac tcttcctgcc tatggctgtg cccatcctcc tgtccctggt ca #agatcttc   1320 ctggagaccc gcaagtcctg gagaaagcct gagaagacag actgagcagg gc #agcacctc   1380 cataggaagc cttcctttct ggccaaggtg ggcggtgtta gattgtgagg ca #cgtacatg   1440 gggcctgccg gaatgactta aatatttgtc tccagtctcc actgttggct ct #ccagcaac   1500 caaagtacaa cactccaaga tgggttcatc ttttcttcct ttcccattca cc #tggctcaa   1560 tcctcctcca ccaccagggg cctcaaaagg cacatcatcc gggtctcctt at #cttgtttg   1620 ataaggctgc tgcctgtctc cctctgtggc aaggactgtt tgttcttttg cc #ccatttct   1680 caacatagca cacttgtgca ctgagaggag ggagcattat gggaaagtcc ct #gccttcca   1740 cacctctctc tagtccctgt gggacagccc tagcccctgc tgtcatgaag gg #gccaggca   1800 ttggtcacct gtgggacctt ctccctcact cccctccctc ctagttggct tt #gtctgtca   1860 ggtgcagtct ggcgggagtc caggaggcag cagctcagga catggtgctg tg #tgtgtgtg   1920 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtc agaggttcca gaaagttcca ga #tttggaat   1980 caaacagtcc tgaattcaaa tccttgtttt tgcacttatt gtctggagag ct #ttggataa   2040 ggtattgaat ctctctgagc ctcagttttt catttgttca aatggcactg at #gatgtctc   2100 ccttacaaga tggttgtgag gagtaaatgt gatcagcatg taaagtgtct gg #cgtgtagt   2160 aggctcttaa taaacactgg ctgaatatga attggaatga tacaaaaaaa aa #aaaaaaaa   2220 actcgagggg gggcccggta cccaattcgc cctatagtga gtcgtattac a  #           2271 <210> SEQ ID NO 53 <211> LENGTH: 2769 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 53 ctgccaccgc cgctcccccc ctcccgctgc cctcgggccg ggctgggtcg ag #ctgcgatg     60 ccctcggact tcatctcatt gctcagcgcg gacctagacc tggaatcgcc ca #agtccctc    120 tactcgcgag attctctgaa gttacaccca tcacagaatt ttcatagagc tg #gactattg    180 gaaggtcagc aaagtaccat tttaaagcat attgtgttag tatgcaaagg ct #ttctctga    240 aatggagttt aaatgggaaa gtatgaaact ttgcaaactc acacaatgta gt #tttctcct    300 aaagagtctg atccttcttt tagagcagct gaatgtttca atgggttttg tt #gctgcgtt    360 tgatgtgctt gttggctgtt ctatctgctt tgagaaacat tgaaaaaaaa at #cattttaa    420 attatgtgtt gagtgttcta ctttagaatt ccagaccttc attttagctt tt #tatacaca    480 aaatagaatg tgaaaaggcc caaacattaa catgaatata aaagagtgac ac #caaacaca    540 taattagttt gaaaattcag aatttagaac atttatatgc tgagcatttc aa #aatatgtt    600 aattatttat tgcaaaagca ttgatgccat tgttattgca ttatacttaa gg #gtaaggaa    660 agccaagtaa tagcgatgat gattgagcta tgcttagaag aaaggggttt ag #aaagttct    720 ggaaatgtta atagagtctg tagattagtt taaacagctt acacttactt ta #gaaaactt    780 ttggctttgt gtgtgtgtgt gttttaacat tttaagacat aaaatagtct tg #tgtttttg    840 tagattttga agtgatttca catatatcat ctttaaatct ttataatttt gg #aattggac    900 agcaaaggta ctaatttccc cattgtagag aagagaaact aaggctgaaa tt #aagaaacc    960 aaaaaaaaaa aaaagtccca cagtagaaga aataatactt tctgcgtttc ag #ttagtgct   1020 ctttatgcaa agcggccacc gtgaaataca catgtaacca gtaacaaaca tt #ttatggtc   1080 tactttaaaa aggccctaaa tatttaagtt tgacttgaga agatttttgc ct #cccttgga   1140 ggtagtccgg gatagtgggg ggcaagtggg ttttgtagtt gagcttaaat tt #ggatacca   1200 acttagctcc ttattagttc tgtgacttcg ggcaagtgat tttacctctc ta #aactttaa   1260 tattcttcct gttaaaatgt agctgtctct taaatttagt gagctaatgt gt #attaaatt   1320 gcctagcata gcgtctagct taataaatgt tagttttttc ctttagtatc ag #ctaaattt   1380 ttttaacagt ataatttaga gcatataaaa tgttctcaaa taaataattg gt #taaatttg   1440 ttttgtacgt gtaagaagcc catagatata cattagtatg aaaatcaaat at #ttttaaaa   1500 ctacaatttg aagtgagaaa tttttagcag tgtttcttag tgaagcagat tt #ctggtttt   1560 tattcatatt caatttgtga ccatgttgta gagacatatt tgtctgtaca aa #cccatctc   1620 atttcctgga caacaaacca tagagtgtca taatcataga gatgatcagg ga #ggagaaag   1680 acatgaagaa aaaaatgagt gttaattatt taagcaaata tttttagtga gc #tgatggaa   1740 atacagacaa ccatttagca tgtttattta catgtttatg cctaaatact tt #cgttaatt   1800 tttcatttta gttaagggga ttctcattta aagtaaggct acccttttag aa #attatata   1860 taaaatataa catgtataag tatcaattct gtcatgccct ttaaaatgtt tt #gagacagg   1920 gtgtcttaaa aaagttgttt gcttctgtaa tgaagtgtct aatctgtgcc ac #ctcattgc   1980 tatttctttt ttattattga cattttaaag aatacataaa agtagagaga at #aaaataag   2040 ccccatatac ccatcaccac agtttaacca gtatctaatc ttgcttcttt ta #tcccttct   2100 tctctctcaa gctggattat tttaaaacaa atatcaaaca tcagatcact tc #atacataa   2160 gtatttctgt attacagtga ctttcttata ttaattattg atataattta at #tgttaata   2220 taattattgt attaattttt aattactctg tatagtttat aaactatttc tt #ggaatatt   2280 ctagtttatt tcagcaaagt ctcttgtgac tctcctgccc tcttttcaag ac #agagtgct   2340 gcaggaggct accgcttcta gtatatagta tatcctataa cctatgttga aa #ttgtaggc   2400 catgctgcta gattggagtt catattgttg ctctgacatt tacttgtatg gc #cttagcct   2460 aataatttaa tcttttttgc ttcaattttc tcatctttaa acgggagcaa ta #atagtgcc   2520 tcatcctagg tttttgtgaa gattatttga ggcaaattcg tgtaaagcat tt #aatttagc   2580 acatgctaag tgctcagtaa atgttagcta aagttaaaaa atttttaaaa cc #taaatgct   2640 agctgtagag tgtttactgt gtgccaagaa ctttacaaat aagaactcct ga #ggtgggca   2700 tatcacttgc ggtcaggagt ttgagaccag cctggcttac atggtgaaac cc #catctcta   2760 ctaaaaaaa                 #                   #                   #       2769 <210> SEQ ID NO 54 <211> LENGTH: 1389 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 54 ctgcaggatt cggcacgagg atctgttgac cacagatgta agaatttatt tc #tggagcct     60 caattctaat ccattgctct acatgtgcac tggtttcctg gggctgccac ag #cagaatac    120 cacagacttg ggggcttaaa caacaaagtg attcctcaca gttctggagg ct #ccaagtct    180 gagatcaagg agtcggcatg tttggttcct cctgaggctt ctctccttgg cc #tgcagatg    240 accacgttct cactgtgttc tcagctggcc cttctctgtg catgtacatc cc #tggtgtct    300 cttcctcctt ttgttgacta taaggatacc agtcctgttg gaccagagcc cc #actgtaaa    360 ggcctcattt taacttaatc cacttaaata acttatttcc acacacagta ag #tctgaggt    420 tgagtctttt gaattctggg ggagacactg cagcccatca cgtatgttga cg #tgtgccac    480 cgccacactg cctggttact gcagagtgtt agtaagtttt gaaattagaa ag #tataagtg    540 ctccagttct gttatttttc aggattatta gggctattct gggtcccttc ca #tctccata    600 tgaatttgag aatcagcttg tttattcctg ctaaaattta gttgggattt tt #tttttttt    660 gagatggagc ctggctgtgt caccaggctg gtgtgcagtg gtgcaatctc gc #ctcactgc    720 aacctccacc tcccgggttc aagtgattct cctgcctcag cctcctgagt ag #ctgggact    780 acatgcgcac gccaccacac tcagctaatt tttgtatttt tagtagagac gg #ggtttcac    840 catgttggcc aggatggtct tgatctcttg acctcgtgat ccgcccacct tg #gcctccca    900 aagtgctggg attacaggca tgagccaccg cgcctggcct cagttgggat tt #tcacaggg    960 ctcgttttga gtctgcagat caatttgggt agtactgaca tcttaaaatt aa #gtctaccg   1020 atccacataa gtgggatgct agttaatatg gacaagtgaa tttattactc ct #ccaaaaaa   1080 ctattttcac aaactttata tcaacatgag aaatcatgtt gtatctttct ac #ctggaggg   1140 agcaagaaag tcaggtggtg tgccttttgt acatgggcaa cccaatttct ta #tctagact   1200 ttcttttgct cgcctggaaa aaaaaaaaaa tgaggtattg caaaaatgtg at #aattgctt   1260 tccttagtat gaaaaactgt taaaatgagt aaagcctgtg ctcaactaga at #tctcacat   1320 ttccgttttg atgtatccgc atttcacctc gtgccgaatt cgatatcaag ct #tatcgata   1380 ccgtcgacc                 #                   #                   #       1389 <210> SEQ ID NO 55 <211> LENGTH: 748 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (15) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (17) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (29) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (32) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (646) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (741) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 55 ggtgcggccg tttanantag ggatccccng gntgcaggaa ttcggcacga gc #ttattgka     60 tgtgctmcag tgacaggcat ctgggacttt tccaatctga agctgttagg aa #caccacca    120 ccatgaatat tcttgtatgt gttttctggc tctggggtgg agttgctggg tc #atggggca    180 ggcacatttt catttttact tcagtaaaaa atgtcyagkg ggccagccac tg #tgcctggc    240 ccaratgact tcttcacaag gagacacata ctgtgtgtat cagtcaggat cc #aaccagga    300 gacaaaccac acagtaattt aaacagcgat tgtttaatat acagaattgt ta #actatgat    360 aggggatttg agtaagagga actggttact aagaaataaa gagaatgcta ac #gaatgtag    420 aaatagactg ggcacaatgg ctcacacctg taatcccagc actttgggag gc #aaggcagg    480 tggatcacga ggtcaagaga tcgagaccat cctggccaac atggtgaaac cc #cgtcccta    540 ctacaaaaag tagctgggcg tggtggcgtg tgcctgtagt cccagctact ca #aggaggct    600 gaggcaggag aatagtttga acccaggagg cagaggttgc ggtganccga ga #tcttgcca    660 ctgcactcca gcctgggtga cagagagaga ctccatctca aaaaaaaaaa aa #aaaaaaaa    720 actcgagggg gggcccggta nccaattc          #                   #            748 <210> SEQ ID NO 56 <211> LENGTH: 4202 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (57) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (58) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (61) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (4137) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 56 accagctcaa agattcatga gtttcatcga gtcactgtga gtggagccca tg #ctggnntg     60 ntgccctctg tgtctgtgca tgcgcgtgtg tgtgtgggcg tgtgtgcatt gc #tgggccag    120 cttgaaggga aggcccgtca tgtccctgca ctctgttttg caagatgcca aa #ccccagtt    180 ctgatggggc tccaacagcc aggctgtggt cctttgacgt tcctcacctg tt #gccaacct    240 atcccgtagt gaactgaaac cccaatgaag acagaactgt gcctggggag at #gcaatgag    300 gtgagggctg aactcatcct tttatatttc ttttcaagat tggatcagag ct #catctcca    360 tccagtcttg tttctatgaa ggcttcaatc tgtttccatg caaatttgct aa #tcagagcc    420 cagagctgct gggtccctca tctccctcat ctattataga ttgacttaca gc #agggagag    480 aatctcttta gctcattcct aatggggttg ggatcacaat atggtctggt cc #aatctgca    540 tcttgttgtg tcccaagacc ctatctcctc cccaacattc ttattgcctt tg #gctcccag    600 taaggaacga attgggggcc agggaggaga acagggggga tcaagaaggg aa #acccaatt    660 ccccctttga aagtgggttc tttgaactat gtgtttgggg gaagttcctc tg #gatactaa    720 tttgaattta tatacctcat gttttggggg tttgacgtat atatatatat at #atatatat    780 atgcatatat atttcataat atttggaagg tttttgatgc tagaaaaatg ga #aacaagag    840 aaccttcaaa aatggtactt agatgggaac tggaggccaa tctttcataa ag #ccagcccc    900 atagctgctt gctgttaggc ctccagccat tttgacattg gggtggatag tc #gattcacc    960 tgcctgtcag tcgattcacc tgcctgtcac ccagttctgt ggatgtgctg gt #gctgagcc   1020 tttgctctct ttccaaatgg ttacagggat gttgatcagc tccaccagag gg #agctctga   1080 tgggaggaat tgctctgcca tccttgtccc tgtgtctcct gtcggcaggc ag #ccattgta   1140 tctcaccagc agaccaggag actggtccca aggttactgc accacagggc aa #tttcctgc   1200 catagttagg aaggaaacac ctgaactaaa tggaagagac atccctgcgg tg #tttaatat   1260 cacacccatg ccctttgtca ggttaccatg tacagagatt acttggagag cc #tcatgccg   1320 tctctacctt cgcacactgg tcaagtatct gctgagcttc ttggccgcaa gg #atgcagaa   1380 ataggctgag ggtccatggg aagaaagaca caatgaggca gtaggaggtg gg #gaagaaaa   1440 gaagacagac tttcaaaatg gaattaggca ctggggagag atcagtttcc cc #acatcagg   1500 gagaagaagg tataggtggg gaagggggtg gccaggagca gaaggaagaa ga #ctcaagat   1560 ggaaagggag ccgctgtgcc tgtggcaata ccacttggag aggtcgactt ca #taccttca   1620 agccttttcc cctgggcttt tgattgtgtc tgtgccccct ttcttgtcct ct #ctgcagat   1680 gcccagtagg ggctacctca tcctcgtgct gttcttgtgt ggctttctgg gc #agtaggga   1740 tcttgaattt cctttctaac actgtgcccg gcaaggcggg gagcattcct ct #gccctttg   1800 tcttgtgcca acctggaaag gtgcagtcta gatttcagtg agaaccctgc ca #gctgagcc   1860 ctgtgcatct actaccttga cacagagtgt tttcccacta gaagctctgc tc #tgctctcc   1920 tggcccaagt aggggattcc atgccttccc tttcatggtc ttagcaccag ca #gcctagtt   1980 tctcccttcc agagtctcca gggatgacaa attggattgg agacaaacct cg #tcagatgc   2040 tcatccccta aaaggttaat tgtgtatttg tggctgcgtg tgcctttgtg tt #ttcattct   2100 cttcccattt ttgtacattt tggtcttctc tgtggtttta tacttggtca aa #agtactcg   2160 tcttggtatt gcactgttgt gtgcatgaga aaactggggg aaggctcact gg #tacaagaa   2220 aggacccctg acccctttcc ttctctgtgg tccccggcat tagattgggg gt #tctgggag   2280 aggcaggtga atgtcctaag tgaattgttc tgtttgtaac tggaatgttt tt #gaagtctt   2340 tggtgttgct ccgtgaaagg acatcgccac ctggtgctca tgaggtgtct tt #gcagaaca   2400 ataaatggca aatgaacaac camaaaattg ttacycttgt tggccttctg ct #gtttgtag   2460 attagtgcac ctatctgtga gggatttggg ttacctccct gagtctgtaa gc #aaccacaa   2520 gccctgccac tgggtggggg aagtccctcc ccaaccactt aaaaacaaat tt #tcccacat   2580 attaccccac cccacacatt tgaccctggc tagactttgt ttgcctaaag ga #acagacca   2640 cattgctggg aaaatgagta agtgaacgtg tgggagaaaa acacttttag aa #tcacgaat   2700 attcactttt aaaggtctct ttgcctggct gcaatatagt gtgtgtttaa at #tatttaca   2760 ggctgttgtt tctcaaataa atgtttaata ttaatcattc ccaaactgac aa #gaacacaa   2820 aaataaaatg caaatacaga gccagctttg tcacccaaat ctgtgtctat tt #ctgatagt   2880 ccatggaatg tggttttctt ggaagccagg gttggtctcc ccacagaccc ca #ggctaagg   2940 tcaccagtta ggaacccagg acttggaagg cagagctgtg agctcttcca tc #agggatct   3000 gactccgcaa aacgacttga tgaatgcaat tggcaaactc ccatgttcgg ac #ttcatatg   3060 catgagccgt tggacagagg gtttcttagt atatacttta atgcatgttt at #gtgcaatc   3120 ttgttagtgg gtatacaagt ttgtgaagaa cttctcattt caataggcag tt #aatgtaat   3180 gcattaaaag cctgggaatt tggggctata tttttccttt ctgactcaat aa #tcttcaaa   3240 gaattcatag gaaagtcagt acttgcagac aagtggttag cttggctaaa at #gtacaaaa   3300 cacccagaac ccacaaaaca ctcagaggtt taggagaatg ttttaatgct ta #agaggcag   3360 gatcaagtga agaggttaca gaaatcagtg tctctggctg ggcagtcaag ag #agcgggct   3420 caaattctgt gactcacttc tctgtgtctc ggttggaaat gaatgggtat cc #tggttccc   3480 accttcccac acgctgtgat acttcaaact ccttgggtga agggcctctt ct #cagcccaa   3540 gatcttgatt gtgaacatta acaaagagaa cagtcatcct ccacagaaga ta #actcatta   3600 atgacatttg attcagtgaa taaatatatc atttaaaaaa atattgtagg gg #gatcatga   3660 aagtagtgga ggtaattaca atcaggagag attggtatta aaattgagca aa #gtcccaac   3720 tctcaccaga tgacaattat gcatcctgct agatgcccca gggctgtcag cc #tggaactg   3780 aaataaatgt gttataagtg gtgctggatg cctttttcag ttcatttgaa aa #catggatt   3840 tgatcatgtc agctcccttt ctgctggaaa aaaaagtagt ttgcataatt gg #tgttaact   3900 actctgtttt gattctacag agtaagtaat actcaaatgt ggtcttactt ta #acttcttg   3960 cctttgttac ccccagaacc atgcagacat tgaaatgtgg tgtgcgtgtg tg #tgtgtgtg   4020 tgtgtgtgtg tgtgtgttca tataacagga ggacaggaaa ggtaaggacc ca #gaacaatg   4080 aagacttatt gaaatgtggg gggggtgtgt gtgtgtgtgt gttcatagaa ca #ggagnaca   4140 ggaaaggtaa tgaccatcca tggaagatga aggggtagta cttagccgga cg #cgtgggtc   4200 ga                   #                   #                   #            4202 <210> SEQ ID NO 57 <211> LENGTH: 854 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (831) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (839) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (844) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (851) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 57 cgaaattact cctcactaaa gttgaacaaa agctggagct cgcgcgcctg ca #ggtcgaca     60 ctagtggatc caaagaattc ggcacgagcg ggatcccggc aaaatgcaga tt #ccccaaaa    120 tttttgtaaa tacagatgac acttatgaag agctccattt aatcgtttat aa #ggtaacaa    180 ctgttttcct tccagcgtta atgattgtgc tgaagtggat ctttcttgca tg #tgtacacg    240 agtgcatgtg caaacctctt aaatgtttct tggaaaagat attggaagtt ct #gattatgg    300 taaaactcaa aatgggtgtt cttccagcgt aataagttta ttttcagctc ct #tttaaaca    360 gttctgttat tagtgaaaga ggaactgttt aagattgtga tttataaacg tg #tgaagtct    420 cacgtgctct caaaccaaag gctgtcagag gttggtgctg cctgttctcg aa #atggctct    480 ggatgggggc cgtagccacg tgtctgtgca tatgctgctt ttgctctgat tt #taaagctg    540 taggcttgct aattccataa ggatcgtatt ttgkttctgt caggacatgg tc #ttgtaagg    600 atatgtactc aggttgtgtt tctaattaaa ggcagttttt gattcaagaa ag #aagacgga    660 gcatgtgcac gtgtttctcc tctttcctgc ctgaggctgt ggagaagttt tc #atttataa    720 aggctcagaa atgatgccgt gggggamcag gaaggagcgg agaactagtc tc #gagagtac    780 ttctagagcg gccgcgggcc catcgatttt ccacccgggt ggggtaccag nt #aagtgtng    840 aagnattccc ntta               #                   #                   #    854 <210> SEQ ID NO 58 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (5) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (39) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (40) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 58 ggcanagctg ggatggggtc agcacccaga agccagccnn ctctgacagc tt #cctctttg     60 gccaagccct gcctctgtac agcctcgagt ggacagccag agggcacgag gg #agcccaga    120 gcccaagatg gagccccagc tggggcctga ggctgccgcc ctccgccctg gc #tggctggc    180 cctgctgctg tgggtctcag ccctgagctg ttctttctcc ttgccagctt ct #tccctttc    240 ttctctggtg ccccaagtca gaaccagcta caattttgga aggactttcc tc #ggtcttga    300 taaatgcaat gcctgcatcg ggacatctat ttgcaagaag ttctttaaag aa #gaaataag    360 atctgacaac tggctggctt cccaccttgg actgcctccc gattccttgc tt #tcttatcc    420 tgcaaattac tcagatgatt ccaaaatctg gcgccctgtg gagatcttta ga #ctggtcag    480 caaatatcaa aacgagatct cagacaggaa aatctgtgcc tctgcatcag cc #ccaaagac    540 ctgcagcatt gagcgtgtcc tgcggaaaac agagaggttc cagaaatggc tg #caggccaa    600 gcgcctcacg ccggacctgg tgcaggactg tcaccagggc cagagagaac ta #aagttcct    660 gtgtatgctg agataacacc agtgaaaaag cctggcatgg agcccagcac tg #agaacttc    720 cagaaagtgt tagccttctc ccaactgtgt tataccaacc acattttcaa at #agtaatca    780 ttaaagaggc ttctgcatca aaccttcaca tgcagctccc atgccacctc ca #gaattcac    840 caacacacag gcccaccagc aacaggtacc tttgcacaat attttttgat ga #caatccaa    900 agccccggct ctttcccacc acactgtggt cccctagatg gggctgttgc tg #agcccacc    960 ccaatcccag atgtgatccc ccctgtgatc tacttcctgg caagattcct cc #agtcctgg   1020 acaggtcttc cctatgagat agaacctgat aaggagctag ggcaattctg ac #aacattac   1080 caaaggccca cataacttct aaattttggt ctggtctgaa ggaaaacctg tt #cttgccct   1140 agtgatggat gaactctctt atctctggct tctagaggga aaaaaaagca ta #cctctttt   1200 actttttaag tacctccatc agagtcatga aatcacctgt caagactatc ta #tcttttat   1260 gtttccattc tggtaagaac tctttaaatg aggacactgc tgattgctgg tg #atgttttt   1320 tgagcaaaca ctcgggggta tggatgaaag ccaatcgcag gtcaaatgac tc #cttgggga   1380 agctacttct cctctattca gatttcacta aaatcttcca agatgaaagc aa #aaaaaaaa   1440 aaaaaaaaaa aaaaa               #                   #                   #  1455 <210> SEQ ID NO 59 <211> LENGTH: 593 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 59 ccacgcttcc ggagctctta cttctccagc aacgctcttc agtacataat aa #gcttaact     60 gataaacaga atatttagaa aggtgagact tgggcttacc attgggttta aa #tcataggg    120 acctagggcg agggttcagg gcttctctgg agcagatatt gtcaagttca tg #gccttagg    180 tagcatgtat ctggtcttaa ctctgattgt agcaaaagtt ctgagaggag ct #gagccctg    240 ttgtggccca ttaaagaaca gggtcctcag gccctgcccg cttcctgtcc ac #tgccccct    300 ccccatcccc agcccagccg agggaatccc gtgggttgct tacctaccta ta #aggtggtt    360 tataagctgc tgtcctggcc actgcattca aattccaatg tgtacttcat ag #tgtaaaaa    420 tttatattat tgtgaggttt tttgtctttt tttttttttt tttttttttg gt #atattgct    480 gtatctactt taacttccag aaataaacgt tatataggaa ccgacaaaaa aa #aaaaaaaa    540 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa #a           593 <210> SEQ ID NO 60 <211> LENGTH: 496 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 60 aattcggctt tcgagcggcc gcccgggcag gtcctgcttt ttgcttataa tt #gacaacat     60 gtgcaaaaat accaaatttg tgtcctgtgc agtatgaaga attcagtgaa ta #ttcattaa    120 tgtattagct tgttttgctc tctgttcata tatggctcta ttcttagaaa ta #taatttga    180 atgtgatctt tcaatagtct gaatatttta caaattatag ctatgtcttg tg #aaaataac    240 ctcaaaaaga aaaatacgac tctgttgtct tacttgatat ttcttgccct ag #taatgtac    300 ttgacattta tgttcctaag cagtgtaagt accagtagaa tttctctgtc aa #actcaatg    360 atcatttagt acttttgtct tctcccatgt gcttgaagga aaaataaagt gt #cactaccg    420 tatttcttgt tttcatcaaa aaataaaaat aatttaaaaa aaaaaaaaaa aa #aaaaaaaa    480 aarrgggsgg cccccc              #                   #                   #   496 <210> SEQ ID NO 61 <211> LENGTH: 1292 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (71) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (697) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1280) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1287) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1291) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 61 aaacctcttc tataggtaaa gctggtacgc ctgcaggtac cggtccggaa tt #cccgggtc     60 gacccacgcg nccggaaaga ggaaacatag aggtgccaaa ggaacaaaga ca #taatgatg    120 tcatccaagc caacaagcca tgctgaagta aatgaaacca tacccaaccc tt #acccacca    180 agcagcttta tggctcctgg atttcaacag cctctgggtt caatcaactt ag #aaaaccaa    240 gctcagggtg ctcagcgtgc tcagccctac ggcatcacat ctccgggaat ct #ttgctagc    300 agtcaaccgg gtcaaggaaa tatacaaatg ataaatccaa gtgtgggaac ag #cagtaatg    360 aactttaaag aagaagcaaa ggcactaggg gtgatccaga tcatggttgg at #tgatgcac    420 attggttttg gaattgtttt gtgtttaata tccttctctt ttagagaagt at #taggtttt    480 gcctctactg ctgktattgg tggataccca ttctggggtg gcctttcttt ta #ttatctct    540 ggctctctct ctgtgtcagc atccaaggag ctttcccgtt gtctggtgaa ag #gcagcctg    600 ggaatgaaca ttggtaggtc tatcttggcc ttcattggag tgattctgct gc #tggtggat    660 atgtgcatca atggggtarc tggccaagac tactggnccg tgctttctgg aa #aaggcatt    720 tcagccacgc tgatgatctt ctccytcttg gagttcttcg tagcttgtgc ca #cagcccat    780 tttgccaacc aagcaaacac cacaaccaat atgtctgtcc tggttattcc aa #atatgtat    840 gaaagcaacc ctgkgacacc agcgtcttct tcagctcctc ccagatgcaa ca #actactca    900 gctaatgccc ctaaaagaaa aaggggtatc agtctaatct catggagaaa aa #ctacttgc    960 aaaaacttct taagaagatg tcttttattg tctacaatga tttctagtct tt #aaaaactg   1020 tgtttgagat ttgtttttag gttggtcgct aatgatggct gtatctccct tc #actgtctc   1080 ttcctacatt accactacta catgctggca aaggtgaagg atcagaggac tg #aaaaatga   1140 ttctgcaact ctcttaaagt tagaaatgtt tctgttcata ttactttttc ct #taataaaa   1200 tgtcattaga aacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagggc gg #ccgctcta   1260 gaggatccaa gcttacgtan gcgtgcntgc na        #                   #        1292 <210> SEQ ID NO 62 <211> LENGTH: 398 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 62 cccccctggc ccccccatta atgccatggt ttgggaggag cckgatcccc tg #aaccccat     60 atttaacctc tactgcccms ggaaatgccc tacattattt ttccctaatt gg #aagtataa    120 ttagagtgat gttggtaggg tagaaaaaga gggagtcact tgatgctttc ag #gttaatca    180 gagctatggg tgctacaggc ttgtctttct aagtgacata ttcttatcta at #tctcagat    240 caggttttga aagmtwtggg ggtcttttta gattttaatc cctactttct tt #atggtaca    300 aatatgtaca aaagaaaaag gtcttatatt cttttacaca aatttataaa ta #aattttga    360 actccttctg tttaaaaaaa aaaaaaaaaa aaaaaaaa       #                   #    398 <210> SEQ ID NO 63 <211> LENGTH: 1202 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (282) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (596) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (607) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (1200) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 63 gaattcggca cgagattaag ttgtgcactt taattgggtg aattgtacat gt #ragttata     60 tatctractg tagttgtwat taaaaaacaa caggaggcca tgtgggctgc ta #ggagtagc    120 aatgtctgty cccagccagc aggtagagac cagggctgga cagagkagta tg #ggctgtgc    180 tgcagattat ttgtggtacc caactgttgc ataaaacagg gtgtgatctc tt #gcattgct    240 atgcatgagt ggattcccag taaattgtgc caggctgcct gnatgatgtg tg #gcttgtgc    300 tttggatcgt aatgcttacc tatgctactt aagttacata ccctgtggcc tt #tgtggcca    360 ggactgtggg ctactacctg kagtgattcg ttaggggaaa ggacccacag cc #tgtgcagg    420 aggaaaaaag catctctgag tacagggtgg atgagctgga tgagctgccg gg #caagagcc    480 acgcacaccc aggtggtgag tcttaaggat aaggtggaat ttgccccata gc #tgtcctgg    540 acagaaactg cccagagaag aatgaatgga ggacataggg ctctgtggtc cc #accntttt    600 ttggganacc tgtgactggt cctgttacca tgtcaactta gccccaaacc ca #tctctgat    660 tgacttggtt gcttattttg gcacattctt gctccacaca gccacataca ta #ctggctgc    720 tcctcsaagg ccaggcagat gcagcagctg ttgggccasc aaagargaar gt #cctggaar    780 gttctggcct gaacgctgca tctgttgtgt gacagccaca actgctcagg ct #tccttgtc    840 tgtgggtgca ctgtggggag gagtgttatg ataagaacat tggctctcag tc #ttccctgg    900 ggagaagttt ggcctcacgt gggatttggg cgttgccttt aggaaggctc tc #tgcatgtc    960 tagttccagt ttgtactggg aagaattaaa aaagtctgcc agcttcttta gt #ttgtcctg   1020 tcttttgtga tgattctttc tgagatcccc tcctatcagc tcaggagtgg ga #ttttctgg   1080 agaaggaaag tgtttttcct gttcctcact gctcaccttg gggcattcag ga #acatgggc   1140 ctgatgaatt tgcttgaagg cagtctgtaa tcccatcact ttgggagcca aa #gaggcggn   1200 ca                   #                   #                   #            1202 <210> SEQ ID NO 64 <211> LENGTH: 1517 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 64 gattacgcca actcgaattt aaccctcact aaagggaaca aaagctggag ct #ccaccgcg     60 gtggcggccg ctctagaact agtggatccc ccgggctgca ggaattcggc ac #gagggagc    120 ccagagccca agatggagcc ccagctgggg cctgaggctg ccgccctccg cc #ctggctgg    180 ctggccctgc tgctgtgggt ctcagccctg agctgttctt tctccttgcc ag #cttcttcc    240 ctttcttctc tggtgcccca agtcagaacc agctacaatt ttggaaggac tt #tcctcggt    300 cttgataaat gcaatgcctg catcgggaca tctatttgca agaagttctt ta #aagaaaga    360 aataagatct gacaactggc tggcttccca ccttgggact gcctcccgat tc #ccttgctt    420 tcttatcctg gcaaattact caggatgatt ccaaaatctg gcgccctgtg ga #gatcttta    480 gactggtcag caaatatcaa aacgagatct cagacaggaa aatctgtgcc tc #tgcatcag    540 ccccaaagac ctgcagcatt gagcgtgtcc tgcggaaaac agagaggttc ca #gaaatggc    600 tgcaggccaa gcgcctcacg ccggacctgg tgcaggactg tcaccagggc ca #gagagaac    660 taaagttcct gtgtatgctg gagataacac cagtgaaaaa gccttggcat gg #agccccag    720 cactgagaac ttccagaaag tgttagcctt ctcccaactg tgttatacca ac #cacatttt    780 caaatagtaa tcattaaaga ggcttctgca tcaaaccttc acatgcagct cc #catgccac    840 cctccagaat tcaccaacac acaggcccac cagcaacagg cttacctttt gc #acaatatt    900 ttttgatgac aatccaaagc cccggctctt tcccaccaca ctgtggtccc ct #agatgggg    960 ctgttgctga gcccacccca atcccagatg tgatccccct gtgatctact tc #tgggccaa   1020 gattctccag tctggacagg tcttccccta tgagatagaa cctgataagg ag #ctagggca   1080 attctgacaa cattaccaaa ggcccacata acttctaaat tttggtctgg tc #tgaaggaa   1140 aacctgttct tgccctagtg atggatgaac tctcttatct ctggcttcta ga #gggaaaaa   1200 aaagcatacc tcttttactt tttaagtacc tccatcagag tcatgaaatc ac #ctgtcaag   1260 actatctatc ttttatgttt ccattctggt aagaactctt taaatgagga ca #ctgctgat   1320 tgctggtgat gttttttgag caaacactcg ggggtatgga tgaaagccaa tc #gcaggtca   1380 aatgactcct tggggaagct acttctcctc tattcagatt tcactaaaat ct #tccaagat   1440 gaaagcaaaa aaaaaaaaaa aaaaaaaaaa actcgagggg gggcccgtac cc #aattcgcc   1500 ctatagtgag tcgtatt              #                   #                   # 1517 <210> SEQ ID NO 65 <211> LENGTH: 526 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (66) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (106) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (484) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 65 ctctgacagc ttcctctttg gccaagccct gcctctgtac agcctcgagt gg #acagccag     60 aggtcnagac tggagcccag agcccaagat ggagccccag ctgggncctg ag #gctgccgc    120 cctccgccct ggctggctgg ccctgctgct gtgggtctca gccctgagct gt #tctttctc    180 cttgccagct tcttcccttt cttctctggt gccccaagtc agaaccagct ac #aattttgg    240 aaggactttc ctcggtcttg ataaatgcaa tgcctgcatc gggacatcta tt #tgcaagaa    300 gttctttaaa gaagaaataa gatctgacaa ctggctggct tcccaccttg gg #actgcctc    360 ccgattccct ttgstttctt atccttgcaa attactccar atgattycca aa #atctggsg    420 sccttgtgga ratcttttaa ctggtcagca awtwtcaaac gaaatctcca aa #caggaaat    480 cttntgcctc ctgcatccac ccccaaagaa cttgcacatt gacgtt    #                526 <210> SEQ ID NO 66 <211> LENGTH: 664 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (31) <223> OTHER INFORMATION: n equals a,t,g, or c <221> NAME/KEY: SITE <222> LOCATION: (63) <223> OTHER INFORMATION: n equals a,t,g, or c <400> SEQUENCE: 66 caggctctca atacggactc actcataggg naaagctggt acgcctgcag gt #accggtcc     60 ggnaattccc gggtcgaccc acgcgtcgcr gagctcttac ttctccagca ac #rctcttca    120 gtacataata agcttaactg ataaacagaa tatttagaaa ggtgagactt gg #gcttacca    180 ttgggtttaa atcataggga cctagggcga gggttcaggg cttctctgga gc #agatattg    240 tcaagttcat ggccttaggt agcatgtatc tggtcttaac tctgattgta gc #aaaagttc    300 tgagaggagc tgagccctgt tgtggcccat taaagaacag ggtcctcagg cc #ctgcccgc    360 ttcctgtcca ctgccccctc cccatcccca gcccagccga gggaatcccg tg #ggttgctt    420 acctacctat aaggtggttt ataagctgct gtcctggcca ctgcattcaa at #tccaatgt    480 gtacttcata gtgtaaaaat ttatattatt gtgaggtttt ttgtcttttt tt #tttttttt    540 ttttttggta tattgctgta tctactttaa cttccagaaa taaacgttat at #rggaaaaa    600 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa #aaaaaaaa    660 aaaa                  #                   #                   #            664 <210> SEQ ID NO 67 <211> LENGTH: 156 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (156) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 67 Met Arg Leu Trp Lys Ala Val Val Val Thr Le #u Ala Phe Met Ser Val   1               5  #                 10  #                 15 Asp Ile Cys Val Thr Thr Ala Ile Tyr Val Ph #e Ser His Leu Asp Arg              20      #             25      #             30 Ser Leu Leu Glu Asp Ile Arg His Phe Asn Il #e Phe Asp Ser Val Leu          35          #         40          #         45 Asp Leu Trp Ala Ala Cys Leu Tyr Arg Ser Cy #s Leu Leu Leu Gly Ser      50              #     55              #     60 His His Trp Cys Gly Gln Glu Gln Cys Ala Gl #y Ala Pro Ala Ala Ala  65                  # 70                  # 75                  # 80 Gly Leu Val Ala Gly His His Pro Arg Val Pr #o Leu Arg Gly His Leu                  85  #                 90  #                 95 Cys His Gly Glu Ala Ala Ala Leu Leu Arg Gl #y Ala Gln Ala His Pro             100       #           105       #           110 Gly Pro Leu Val Leu Gly Pro Val Arg Val As #p Val His Phe Thr Arg         115           #       120           #       125 Arg Ile Leu Pro Ala Leu Val Ala Ala Val Hi #s Arg Ala Ala Arg His     130               #   135               #   140 Pro Gly Pro Gly Ala Arg Gly Gly His Arg Gl #y Xaa 145                 1 #50                 1 #55 <210> SEQ ID NO 68 <211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (70) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 68 Met Ala Ala Arg His Leu Pro Gly Phe His Th #r Tyr Thr Asn Leu Leu   1               5  #                 10  #                 15 Phe Leu Leu Leu Pro Ser Leu Leu Met Gly Ty #r Ser Glu Ser Pro Pro              20      #             25      #             30 Pro Ile Thr Asp Ser Trp Ala Pro Phe Ile Se #r Leu Thr His His Val          35          #         40          #         45 Leu Ser Gln Ser Gln Ser Pro Leu Ser Ser As #n Cys Trp Ile Cys Leu      50              #     55              #     60 Ser Thr His Thr Gln Xaa  65                  # 70 <210> SEQ ID NO 69 <211> LENGTH: 502 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (502) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 69 Met Trp Lys Leu Trp Arg Ala Glu Glu Gly Al #a Ala Ala Leu Gly Gly   1               5  #                 10  #                 15 Ala Leu Phe Leu Leu Leu Phe Ala Leu Gly Va #l Arg Gln Leu Leu Lys              20      #             25      #             30 Gln Arg Arg Pro Met Gly Phe Pro Pro Gly Pr #o Pro Gly Leu Pro Phe          35          #         40          #         45 Ile Gly Asn Ile Tyr Ser Leu Ala Ala Ser Se #r Glu Leu Pro His Val      50              #     55              #     60 Tyr Met Arg Lys Gln Ser Gln Val Tyr Gly Gl #u Ile Phe Ser Leu Asp  65                  # 70                  # 75                  # 80 Leu Gly Gly Ile Ser Thr Val Val Leu Asn Gl #y Tyr Asp Val Val Lys                  85  #                 90  #                 95 Glu Cys Leu Val His Gln Ser Glu Ile Phe Al #a Asp Arg Pro Cys Leu             100       #           105       #           110 Pro Leu Phe Met Lys Met Thr Lys Met Gly Gl #y Leu Leu Asn Ser Arg         115           #       120           #       125 Tyr Gly Arg Gly Trp Val Asp His Arg Arg Le #u Ala Val Asn Ser Phe     130               #   135               #   140 Arg Tyr Phe Gly Tyr Gly Gln Lys Ser Phe Gl #u Ser Lys Ile Leu Glu 145                 1 #50                 1 #55                 1 #60 Glu Thr Lys Phe Phe Asn Asp Ala Ile Glu Th #r Tyr Lys Gly Arg Pro                 165   #               170   #               175 Phe Asp Phe Lys Gln Leu Ile Thr Asn Ala Va #l Ser Asn Ile Thr Asn             180       #           185       #           190 Leu Ile Ile Phe Gly Glu Arg Phe Thr Tyr Gl #u Asp Thr Asp Phe Gln         195           #       200           #       205 His Met Ile Glu Leu Phe Ser Glu Asn Val Gl #u Leu Ala Ala Ser Ala     210               #   215               #   220 Ser Val Phe Leu Tyr Asn Ala Phe Pro Trp Il #e Gly Ile Leu Pro Phe 225                 2 #30                 2 #35                 2 #40 Gly Lys His Gln Gln Leu Phe Arg Asn Ala Al #a Val Val Tyr Asp Phe                 245   #               250   #               255 Leu Ser Arg Leu Ile Glu Lys Ala Ser Val As #n Arg Lys Pro Gln Leu             260       #           265       #           270 Pro Gln His Phe Val Asp Ala Tyr Leu Asp Gl #u Met Asp Gln Gly Lys         275           #       280           #       285 Asn Asp Pro Ser Ser Thr Phe Ser Lys Glu As #n Leu Ile Phe Ser Val     290               #   295               #   300 Gly Glu Leu Ile Ile Ala Gly Thr Glu Thr Th #r Thr Asn Val Leu Arg 305                 3 #10                 3 #15                 3 #20 Trp Ala Ile Leu Phe Met Ala Leu Tyr Pro As #n Ile Gln Gly Gln Val                 325   #               330   #               335 Gln Lys Glu Ile Asp Leu Ile Met Gly Pro As #n Gly Lys Pro Ser Trp             340       #           345       #           350 Asp Asp Lys Cys Lys Met Pro Tyr Thr Glu Al #a Val Leu His Glu Val         355           #       360           #       365 Leu Arg Phe Cys Asn Ile Val Pro Leu Gly Il #e Phe His Ala Thr Ser     370               #   375               #   380 Glu Asp Ala Val Val Arg Gly Tyr Ser Ile Pr #o Lys Gly Thr Thr Val 385                 3 #90                 3 #95                 4 #00 Ile Thr Asn Leu Tyr Ser Val His Phe Asp Gl #u Lys Tyr Trp Arg Asp                 405   #               410   #               415 Pro Glu Val Phe His Pro Glu Arg Phe Leu As #p Ser Ser Gly Tyr Phe             420       #           425       #           430 Ala Lys Lys Glu Ala Leu Val Pro Phe Ser Le #u Gly Arg Arg His Cys         435           #       440           #       445 Leu Gly Glu His Leu Ala Arg Met Glu Met Ph #e Leu Phe Phe Thr Ala     450               #   455               #   460 Leu Leu Gln Arg Phe His Leu His Phe Pro Hi #s Glu Leu Val Pro Asp 465                 4 #70                 4 #75                 4 #80 Leu Lys Pro Arg Leu Gly Met Thr Leu Gln Pr #o Gln Pro Tyr Leu Ile                 485   #               490   #               495 Cys Ala Glu Arg Arg Xaa             500 <210> SEQ ID NO 70 <211> LENGTH: 189 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (85) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (104) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (164) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (189) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 70 Met Arg Pro Ala Phe Ala Leu Cys Leu Leu Tr #p Gln Ala Leu Trp Pro   1               5  #                 10  #                 15 Gly Pro Gly Gly Gly Glu His Pro Thr Ala As #p Arg Ala Gly Cys Ser              20      #             25      #             30 Ala Ser Gly Ala Cys Tyr Ser Leu His His Al #a Thr Met Lys Arg Gln          35          #         40          #         45 Ala Ala Glu Glu Ala Cys Ile Leu Arg Gly Gl #y Ala Leu Ser Thr Val      50              #     55              #     60 Arg Ala Gly Ala Glu Leu Arg Ala Val Leu Al #a Leu Leu Arg Ala Gly  65                  # 70                  # 75                  # 80 Pro Gly Pro Gly Xaa Gly Ser Lys Asp Leu Le #u Phe Trp Val Ala Leu                  85  #                 90  #                 95 Glu Arg Arg Arg Ser His Cys Xaa Leu Glu As #n Glu Pro Leu Arg Gly             100       #           105       #           110 Phe Ser Trp Leu Ser Ser Asp Pro Gly Gly Le #u Glu Ser Asp Thr Leu         115           #       120           #       125 Gln Trp Val Glu Glu Pro Gln Arg Ser Cys Th #r Ala Arg Arg Trp Val     130               #   135               #   140 Leu Pro Gly His Arg Trp Gly Arg Ala Arg Se #r Trp Lys Glu Met Arg 145                 1 #50                 1 #55                 1 #60 Cys His Leu Xaa Ala Asn Ala Thr Cys Ala Se #r Thr Ser Leu Arg Ser                 165   #               170   #               175 Cys Val Leu Arg Arg Ala Pro Gly Pro Pro Le #u Thr Xaa             180       #           185 <210> SEQ ID NO 71 <211> LENGTH: 486 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 71 Met Gln Pro Ser Gly Leu Glu Gly Pro Gly Th #r Phe Gly Arg Trp Pro   1               5  #                 10  #                 15 Leu Leu Ser Leu Leu Leu Leu Leu Leu Leu Le #u Gln Pro Val Thr Cys              20      #             25      #             30 Ala Tyr Thr Thr Pro Gly Pro Pro Arg Ala Le #u Thr Thr Leu Gly Ala          35          #         40          #         45 Pro Arg Ala His Thr Met Pro Gly Thr Tyr Al #a Pro Ser Thr Thr Leu      50              #     55              #     60 Ser Ser Pro Ser Thr Gln Gly Leu Gln Glu Gl #n Ala Arg Ala Leu Met  65                  # 70                  # 75                  # 80 Arg Asp Phe Pro Leu Val Asp Gly His Asn As #p Leu Pro Leu Val Leu                  85  #                 90  #                 95 Arg Gln Val Tyr Gln Lys Gly Leu Gln Asp Va #l Asn Leu Arg Asn Phe             100       #           105       #           110 Ser Tyr Gly Gln Thr Ser Leu Asp Arg Leu Ar #g Asp Gly Leu Val Gly         115           #       120           #       125 Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gl #n Thr Gln Asp Arg Asp     130               #   135               #   140 Ala Leu Arg Leu Thr Leu Glu Gln Ile Asp Le #u Ile Arg Arg Met Cys 145                 1 #50                 1 #55                 1 #60 Ala Ser Tyr Ser Glu Leu Glu Leu Val Thr Se #r Ala Lys Ala Leu Asn                 165   #               170   #               175 Asp Thr Gln Lys Leu Ala Cys Leu Ile Gly Va #l Glu Gly Gly His Ser             180       #           185       #           190 Leu Asp Asn Ser Leu Ser Ile Leu Arg Thr Ph #e Tyr Met Leu Gly Val         195           #       200           #       205 Arg Tyr Leu Thr Leu Thr His Thr Cys Asn Th #r Pro Trp Ala Glu Ser     210               #   215               #   220 Ser Ala Lys Gly Val His Ser Phe Tyr Asn As #n Ile Ser Gly Leu Thr 225                 2 #30                 2 #35                 2 #40 Asp Phe Gly Glu Lys Val Val Ala Glu Met As #n Arg Leu Gly Met Met                 245   #               250   #               255 Val Asp Leu Ser His Val Ser Asp Ala Val Al #a Arg Arg Ala Leu Glu             260       #           265       #           270 Val Ser Gln Ala Pro Val Ile Phe Ser His Se #r Ala Ala Arg Gly Val         275           #       280           #       285 Cys Asn Ser Ala Arg Asn Val Pro Asp Asp Il #e Leu Gln Leu Leu Lys     290               #   295               #   300 Lys Asn Gly Gly Val Val Met Val Ser Leu Se #r Met Gly Val Ile Gln 305                 3 #10                 3 #15                 3 #20 Cys Asn Pro Ser Ala Asn Val Ser Thr Val Al #a Asp His Phe Asp His                 325   #               330   #               335 Ile Lys Ala Val Ile Gly Ser Lys Phe Ile Gl #y Ile Gly Gly Asp Tyr             340       #           345       #           350 Asp Gly Ala Gly Lys Phe Pro Gln Gly Leu Gl #u Asp Val Ser Thr Tyr         355           #       360           #       365 Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gl #y Trp Ser Glu Glu Glu     370               #   375               #   380 Leu Gln Gly Val Leu Arg Gly Asn Leu Leu Ar #g Val Phe Arg Gln Val 385                 3 #90                 3 #95                 4 #00 Glu Lys Val Gln Glu Glu Asn Lys Trp Gln Se #r Pro Leu Glu Asp Lys                 405   #               410   #               415 Phe Pro Asp Glu Gln Leu Ser Ser Ser Cys Hi #s Ser Asp Leu Ser Arg             420       #           425       #           430 Leu Arg Gln Arg Gln Ser Leu Thr Ser Gly Gl #n Glu Leu Thr Glu Ile         435           #       440           #       445 Pro Ile His Trp Thr Ala Lys Leu Pro Ala Ly #s Trp Ser Val Ser Glu     450               #   455               #   460 Ser Ser Pro His Met Ala Pro Val Leu Ala Va #l Val Ala Thr Phe Pro 465                 4 #70                 4 #75                 4 #80 Val Leu Ile Leu Trp Leu                 485 <210> SEQ ID NO 72 <211> LENGTH: 88 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (88) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 72 Met Val Ala Ser Gly Trp Leu Leu Leu Ala Gl #n Ala Ser Phe Leu Pro   1               5  #                 10  #                 15 Leu Ala Pro Pro Gly Ala Leu Gly Ala Gly Cy #s Trp Met Asp Gly Arg              20      #             25      #             30 Pro Leu Ala Pro Pro Gly Ala Leu Gly Ala Gl #y Cys Trp Met Gly Gly          35          #         40          #         45 Arg Pro Leu Ala Pro Pro Gly Ala Leu Gly Al #a Gly Cys Trp Met Gly      50              #     55              #     60 Gly Arg His Gly Ala Pro Leu Leu Gly Cys Le #u Cys Pro Ser Gly Leu  65                  # 70                  # 75                  # 80 Cys Ser Ser Tyr Val Cys Leu Xaa                  85 <210> SEQ ID NO 73 <211> LENGTH: 299 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (167) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 73 Met Met Ser Ser Lys Pro Thr Ser His Ala Gl #u Val Asn Glu Thr Ile   1               5  #                 10  #                 15 Pro Asn Pro Tyr Pro Pro Ser Ser Phe Met Al #a Pro Gly Phe Gln Gln              20      #             25      #             30 Pro Leu Gly Ser Ile Asn Leu Glu Asn Gln Al #a Gln Gly Ala Gln Arg          35          #         40          #         45 Ala Gln Pro Tyr Gly Ile Thr Ser Pro Gly Il #e Phe Ala Ser Ser Gln      50              #     55              #     60 Pro Gly Gln Gly Asn Ile Gln Met Ile Asn Pr #o Ser Val Gly Thr Ala  65                  # 70                  # 75                  # 80 Val Met Asn Phe Lys Glu Glu Ala Lys Ala Le #u Gly Val Ile Gln Ile                  85  #                 90  #                 95 Met Val Gly Leu Met His Ile Gly Phe Gly Il #e Val Leu Cys Leu Ile             100       #           105       #           110 Ser Phe Ser Phe Arg Glu Val Leu Gly Phe Al #a Ser Thr Ala Val Ile         115           #       120           #       125 Gly Gly Tyr Pro Phe Trp Gly Gly Leu Ser Ph #e Ile Ile Ser Gly Ser     130               #   135               #   140 Leu Ser Val Ser Ala Ser Lys Glu Leu Ser Ar #g Cys Leu Val Lys Gly 145                 1 #50                 1 #55                 1 #60 Ser Leu Gly Met Asn Ile Xaa Ser Ser Ile Le #u Ala Phe Ile Gly Val                 165   #               170   #               175 Ile Leu Leu Leu Val Asp Met Cys Ile Asn Gl #y Val Ala Gly Gln Asp             180       #           185       #           190 Tyr Trp Ala Val Leu Ser Gly Lys Gly Ile Se #r Ala Thr Leu Met Ile         195           #       200           #       205 Phe Ser Leu Leu Glu Phe Phe Val Ala Cys Al #a Thr Ala His Phe Ala     210               #   215               #   220 Asn Gln Ala Asn Thr Thr Thr Asn Met Ser Va #l Leu Val Ile Pro Asn 225                 2 #30                 2 #35                 2 #40 Met Tyr Glu Ser Asn Pro Val Thr Pro Ala Se #r Ser Ser Ala Pro Pro                 245   #               250   #               255 Arg Cys Asn Asn Tyr Ser Ala Asn Ala Pro Ly #s Arg Lys Arg Gly Ile             260       #           265       #           270 Ser Leu Ile Ser Trp Arg Lys Thr Thr Cys Ly #s Asn Phe Leu Arg Arg         275           #       280           #       285 Cys Leu Leu Leu Ser Thr Met Ile Ser Ser Le #u     290               #   295 <210> SEQ ID NO 74 <211> LENGTH: 48 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (48) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 74 Met Ala Leu His Pro Gly Ser Ser His Leu Le #u Val Ala Val Pro Val   1               5  #                 10  #                 15 Ser Trp Phe Leu Phe Cys Ile Pro Gly Ile Se #r Phe Ile Thr Leu Ser              20      #             25      #             30 Trp Ser Tyr Gln Glu Ser Pro Val Ser Phe Le #u Ser Val Glu Gly Xaa          35          #         40          #         45 <210> SEQ ID NO 75 <211> LENGTH: 44 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (44) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 75 Met Tyr Ser Leu Phe Leu Thr Cys Ile Phe Pr #o Phe Thr Leu Cys His   1               5  #                 10  #                 15 Lys Lys Ile Leu Met Val Ile His Asp Phe Th #r Gly Pro Val His Val              20      #             25      #             30 Phe Pro Glu Lys Thr Val Leu Glu Trp Asn Ty #r Xaa          35          #         40 <210> SEQ ID NO 76 <211> LENGTH: 140 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 76 Met Cys Ala Met Tyr Leu Met Ile Lys Ala Ph #e Leu Pro Lys Met Leu   1               5  #                 10  #                 15 Ala Gln Lys Ser Gly Asn Ile Ile Asn Met Se #r Ser Val Ala Ser Ser              20      #             25      #             30 Val Lys Gly Val Val Asn Arg Cys Val Tyr Se #r Thr Thr Lys Ala Ala          35          #         40          #         45 Val Ile Gly Leu Thr Lys Ser Val Ala Ala As #p Phe Ile Gln Gln Gly      50              #     55              #     60 Ile Arg Cys Asn Cys Val Cys Pro Gly Thr Va #l Asp Thr Pro Ser Leu  65                  # 70                  # 75                  # 80 Gln Glu Arg Ile Gln Ala Arg Gly Asn Pro Gl #u Glu Ala Arg Asn Asp                  85  #                 90  #                 95 Phe Leu Lys Arg Gln Lys Thr Gly Arg Phe Al #a Thr Ala Glu Glu Ile             100       #           105       #           110 Ala Met Leu Cys Val Tyr Leu Ala Ser Asp Gl #u Ser Ala Tyr Val Thr         115           #       120           #       125 Gly Asn Pro Val Ile Ile Asp Gly Gly Trp Se #r Leu     130               #   135               #   140 <210> SEQ ID NO 77 <211> LENGTH: 153 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (153) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 77 Met Leu Val Val Cys Leu Leu Leu Ala Thr Gl #y Phe Cys Leu Phe Arg   1               5  #                 10  #                 15 Gly Leu Ile Ala Leu Asp Cys Pro Ser Glu Le #u Cys Arg Leu Tyr Thr              20      #             25      #             30 Gln Phe Gln Glu Pro Tyr Leu Lys Asp Pro Al #a Ala Tyr Pro Lys Ile          35          #         40          #         45 Gln Met Leu Ala Tyr Met Phe Tyr Ser Val Pr #o Tyr Phe Val Thr Ala      50              #     55              #     60 Leu Tyr Gly Leu Val Val Pro Gly Cys Ser Tr #p Met Pro Asp Ile Thr  65                  # 70                  # 75                  # 80 Leu Ile His Ala Gly Gly Leu Ala Gln Ala Gl #n Phe Ser His Ile Gly                  85  #                 90  #                 95 Ala Ser Leu His Ala Arg Thr Ala Tyr Val Ty #r Arg Val Pro Glu Glu             100       #           105       #           110 Ala Lys Ile Leu Phe Leu Ala Leu Asn Ile Al #a Tyr Gly Val Leu Pro         115           #       120           #       125 Gln Leu Leu Ala Tyr Arg Cys Ile Tyr Lys Pr #o Glu Phe Phe Ile Lys     130               #   135               #   140 Thr Lys Ala Glu Glu Lys Val Glu Xaa 145                 1 #50 <210> SEQ ID NO 78 <211> LENGTH: 180 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (48) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (180) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 78 Met Ala Ala Ala Ser Ala Gly Ala Thr Arg Le #u Leu Leu Leu Leu Leu   1               5  #                 10  #                 15 Met Ala Val Ala Ala Pro Ser Arg Ala Arg Gl #y Ser Gly Cys Arg Ala              20      #             25      #             30 Gly Thr Gly Ala Arg Gly Ala Gly Ala Glu Gl #y Arg Glu Gly Glu Xaa          35          #         40          #         45 Pro Val Ser Ser Ala Ile Pro Arg Arg Val Cy #s Trp Ser Leu Leu Ser      50              #     55              #     60 Pro Arg Pro Thr Arg Pro Pro Gly Pro Ala Pr #o Cys Pro Leu Pro Ser  65                  # 70                  # 75                  # 80 Ala Gly Arg Gly Ala Ala Gly Leu Gly Pro Le #u Ala Gln Gln Pro Val                  85  #                 90  #                 95 Ser Pro Ala Pro Ala Ser Pro Met Ala Pro Cy #s Ser Pro Arg Gly Phe             100       #           105       #           110 Pro Pro Ala His Gly Val Glu Pro Glu Ile Le #u Ala Thr Met Pro Val         115           #       120           #       125 Leu Thr Ser His Pro Pro Thr Pro Ser Pro Cy #s Ser Leu Gly Thr Cys     130               #   135               #   140 Arg Leu Leu Ser Ser Leu Cys Ala Phe Val Pr #o Gly Gly Leu Thr Leu 145                 1 #50                 1 #55                 1 #60 Leu Ser Leu Ala Gly Leu Gly Gly Pro Val Gl #n Ala Pro Ala Ala Pro                 165   #               170   #               175 Pro Ser Leu Xaa             180 <210> SEQ ID NO 79 <211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (70) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 79 Met Leu Met Gly Ser Ile Leu Tyr Val Leu Ph #e Cys Val Trp Leu Leu   1               5  #                 10  #                 15 Gln Cys Ile Phe Glu Ile Tyr Pro His Cys Cy #s Val Tyr Pro Lys Cys              20      #             25      #             30 Val Leu Phe His Cys Gln Ile Met Phe Cys Ty #r Met Asn Ile Leu Gln          35          #         40          #         45 Asn Ile Cys Leu Phe Ile Tyr Trp Trp Ile Ph #e Ala Phe Val Pro Val      50              #     55              #     60 Trp Gly Tyr Tyr Glu Xaa  65                  # 70 <210> SEQ ID NO 80 <211> LENGTH: 191 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (191) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 80 Met Arg Ala Cys Pro Trp Ala Gln Val Pro Le #u Tyr Leu Leu Leu Asp   1               5  #                 10  #                 15 Gly His Leu Ala Val Ser Gln Ala Gly Val Me #t Ala Gly Val Ser Gly              20      #             25      #             30 Gly Arg Gly Gly Arg Arg Leu Arg Gly Pro Il #e Thr Ser Arg Val Ile          35          #         40          #         45 Thr Ser Cys Gln Gln Pro Gly Val Gly Val Tr #p Val Ser Leu Arg Pro      50              #     55              #     60 Glu Leu Leu Asn Leu Glu Ser Leu Gly Val Al #a Ala Lys Gly Val Tyr  65                  # 70                  # 75                  # 80 Asp Lys His Val Ser Leu Asp Ile Ser Gly Gl #u Arg Ser Gly Ala Leu                  85  #                 90  #                 95 Val Thr Phe Ser Lys Gly Cys Trp Ala Ser Gl #u Gln Ser Pro Pro Met             100       #           105       #           110 Ser Gln Pro Leu Gln Gly Pro Ser Leu Ser Le #u His Pro Arg Pro Ser         115           #       120           #       125 Ala Ala Leu Val Met Ser Arg Arg Lys Val Le #u Gly Cys Ala Gln Ser     130               #   135               #   140 Gln Glu Ser Lys Ile Cys Gln Ala Lys Ala Pr #o Gly Lys Ser Arg Arg 145                 1 #50                 1 #55                 1 #60 Ser Leu Gly Trp Pro Pro Gly Cys Gly Ala Al #a Arg Ala Lys Thr Val                 165   #               170   #               175 Asn Thr Ala Leu Gln Leu Ser Glu Pro Gln Ph #e Ser Asn Leu Xaa             180       #           185       #           190 <210> SEQ ID NO 81 <211> LENGTH: 166 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (127) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (166) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 81 Met Cys Leu Ser Leu Leu Ala Ala Leu Ala Cy #s Ser Ala Gly Asp Thr   1               5  #                 10  #                 15 Trp Ala Ser Glu Val Gly Pro Val Leu Ser Ly #s Ser Ser Pro Arg Leu              20      #             25      #             30 Ile Thr Thr Trp Glu Lys Val Pro Val Gly Th #r Asn Gly Gly Val Thr          35          #         40          #         45 Val Val Gly Leu Val Ser Ser Leu Leu Gly Gl #y Thr Phe Val Gly Ile      50              #     55              #     60 Ala Tyr Phe Leu Thr Gln Leu Ile Phe Val As #n Asp Leu Asp Ile Ser  65                  # 70                  # 75                  # 80 Ala Pro Gln Trp Pro Ile Ile Ala Phe Gly Gl #y Leu Ala Gly Leu Leu                  85  #                 90  #                 95 Gly Ser Ile Val Asp Ser Tyr Leu Gly Ala Th #r Met Gln Tyr Thr Gly             100       #           105       #           110 Leu Asp Glu Ser Thr Gly Met Val Val Asn Se #r Pro Thr Asn Xaa Ala         115           #       120           #       125 Arg His Ile Ala Gly Lys Pro Ile Leu Asp As #n Asn Ala Val Asn Leu     130               #   135               #   140 Phe Ser Ser Val Leu Ile Ala Leu Leu Leu Pr #o Thr Ala Ala Trp Gly 145                 1 #50                 1 #55                 1 #60 Phe Trp Pro Arg Gly Xaa                 165 <210> SEQ ID NO 82 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (42) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 82 Met Cys Gly Leu Val Ile Leu Trp Pro Cys Il #e Met Thr Leu Phe Ser   1               5  #                 10  #                 15 Ser Leu Ser Thr Gly Asp Val Leu Leu Pro Cy #s Lys Ile Leu Val Gly              20      #             25      #             30 Leu Arg Val Phe Ile Gly Ala Arg Val Xaa          35          #         40 <210> SEQ ID NO 83 <211> LENGTH: 49 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (49) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 83 Met Cys Phe Pro Ala Cys Leu Cys Ser Pro Le #u Thr Cys Leu Leu Ser   1               5  #                 10  #                 15 Val Trp Lys Pro Gly Leu Ala His Ala Val Va #l His Cys Met Leu Glu              20      #             25      #             30 Pro Val Glu Phe Ala Arg Val Val Gln Tyr Gl #u Ala Gly His Val Leu          35          #         40          #         45 Xaa <210> SEQ ID NO 84 <211> LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (57) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 84 Met Leu Ile Ala Lys Leu Pro Val Leu Glu Se #r Ile Cys Phe Phe Met   1               5  #                 10  #                 15 Leu Phe Leu Asn Pro Leu Val Ile Leu Leu Se #r Leu Asn Asn Ala Leu              20      #             25      #             30 Pro Leu Val Phe His Pro His Ser Glu Phe Le #u Glu Asp His Asn Arg          35          #         40          #         45 Gly Asp Thr Leu Pro Ser Ile Val Xaa      50              #     55 <210> SEQ ID NO 85 <211> LENGTH: 43 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (43) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 85 Met Leu Val Ala Thr Ala Val Cys Cys Tyr Le #u Phe Trp Leu Ile Ala   1               5  #                 10  #                 15 Ile Leu Ala Gln Leu Asn Pro Leu Phe Gly Pr #o Gln Leu Lys Asn Glu              20      #             25      #             30 Thr Ile Trp Tyr Val Arg Phe Leu Trp Glu Xa #a          35          #         40 <210> SEQ ID NO 86 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (41) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 86 Met Leu Leu Leu Trp Ala Phe Ser Gly Val Cy #s Ala Val Pro Ala Arg   1               5  #                 10  #                 15 Ala Thr Pro Val Pro Ser Ser Phe Cys Pro Gl #n Gly Pro Ser Leu Cys              20      #             25      #             30 Pro Lys Gln Pro Ala Ser Leu Ala Xaa          35          #         40 <210> SEQ ID NO 87 <211> LENGTH: 74 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (74) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 87 Met His Ala Tyr Ala Cys Val Cys Ala Cys Me #t Leu Val Cys Val Cys   1               5  #                 10  #                 15 Val Cys Val Cys Arg Ala Leu Val Ile Pro Th #r Glu Gln Arg His Arg              20      #             25      #             30 Arg Val Ala His Gly Arg Thr Ser Asp Ser Th #r Leu Pro Cys Thr Val          35          #         40          #         45 Lys Ile Trp Pro Ser Glu Arg Gly Asp Gly Ar #g Gly Glu Arg Gly Glu      50              #     55              #     60 Arg Arg Arg Gly Thr Asp Trp Arg Gly Xaa  65                  # 70 <210> SEQ ID NO 88 <211> LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (34) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (47) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 88 Met His His Pro Asn Leu Cys Leu His Phe Hi #s Ala Ala Phe Ser Leu   1               5  #                 10  #                 15 Cys Val His Gly Cys Leu Cys Val Gln Phe Ph #e Pro Phe Tyr Lys Asp              20      #             25      #             30 Thr Xaa His Ile Gly Leu Glu Pro Thr Leu Me #t Thr Ser Ser Xaa          35          #         40          #         45 <210> SEQ ID NO 89 <211> LENGTH: 63 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (63) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 89 Met Leu Phe Leu Asn Val Ile Leu Phe Ser Le #u Thr Val Phe Thr Leu   1               5  #                 10  #                 15 Ile Ser Thr Ala His Thr Leu Asp Arg Ala Va #l Arg Ser Asp Trp Leu              20      #             25      #             30 Leu Leu Val Leu Ile Tyr Ala Cys Leu Glu Gl #u Leu Ile Pro Glu Leu          35          #         40          #         45 Ile Phe Asn Leu Tyr Cys Gln Gly Asn Ala Th #r Leu Phe Phe Xaa      50              #     55              #     60 <210> SEQ ID NO 90 <211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (20) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (70) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 90 Met Leu Leu Lys Leu His Thr Leu Trp Pro Le #u Trp Pro Gly Leu Trp   1               5  #                 10  #                 15 Ala Thr Thr Xaa Ser Asp Ser Leu Gly Glu Ar #g Thr His Ser Leu Cys              20      #             25      #             30 Arg Arg Lys Lys Ala Ser Leu Ser Thr Gly Tr #p Met Ser Trp Met Ser          35          #         40          #         45 Cys Arg Ala Arg Ala Thr His Thr Gln Val Va #l Ser Leu Lys Asp Lys      50              #     55              #     60 Val Glu Phe Ala Pro Xaa  65                  # 70 <210> SEQ ID NO 91 <211> LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (57) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 91 Met Lys Glu Ser Arg Lys Met Leu Trp Val Ph #e Lys Met Leu Phe Phe   1               5  #                 10  #                 15 Lys Ile Val Leu Trp Val Asn Leu Leu Ser Al #a Ala Leu Ser Cys Ile              20      #             25      #             30 Gln Lys Gln Met Leu Gly Ile Ala Pro Gln Ly #s Cys Val Pro Lys Leu          35          #         40          #         45 Cys Phe Gln Leu Tyr Ile Met Arg Xaa      50              #     55 <210> SEQ ID NO 92 <211> LENGTH: 68 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (68) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 92 Met Tyr Phe Leu Leu Ser Val Thr Ser Glu Se #r Val Trp Arg Ser Trp   1               5  #                 10  #                 15 Thr Leu Thr Phe His Ser Phe Ala Ile Leu Se #r Leu Arg Cys Trp Thr              20      #             25      #             30 Ser Leu Leu Leu Leu Ile Pro Leu Thr Ser Cy #s Asn Phe Ser Ser Pro          35          #         40          #         45 Ser Trp Arg Met Thr Ala Ser Gln Val Pro Se #r Lys Arg Lys Ala Ser      50              #     55              #     60 Met Thr Leu Xaa  65 <210> SEQ ID NO 93 <211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (45) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 93 Met Lys Gly Trp Pro Val Phe Leu Leu Val Gl #n Ala Val Thr Phe Leu   1               5  #                 10  #                 15 Ser Val Ala Gln Ser Gly Ala Met Ala Cys Al #a Ala Ser Gly Val Val              20      #             25      #             30 Tyr Ser Val Asp Val Pro Ala Cys Ser Ser Ar #g Ser Xaa          35          #         40          #         45 <210> SEQ ID NO 94 <211> LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (55) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 94 Met Val Leu Ser Pro Trp Ala Cys Leu Phe Va #l Val Phe Phe Pro Tyr   1               5  #                 10  #                 15 Ile Gln Ser Ser Leu Arg Ser Asp Lys His Le #u Gln Leu Ser Asn Ile              20      #             25      #             30 Leu Pro Thr Pro Ser His His Ile His Leu Pr #o Ala Ser Ile Cys Ile          35          #         40          #         45 Gln Leu Arg Ala Gly Asn Xaa      50              #     55 <210> SEQ ID NO 95 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (41) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 95 Met Cys Glu Tyr Val Leu Leu Leu Tyr Ile Va #l Leu Leu Cys Asn Arg   1               5  #                 10  #                 15 Ser Tyr Ala Val Phe Thr Gln Cys Val Leu Ar #g Ser Ser Pro Ile Asp              20      #             25      #             30 Ser Ser Arg Asn Ala Val Leu Leu Xaa          35          #         40 <210> SEQ ID NO 96 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (41) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 96 Met Thr Thr Pro Gly Leu Leu Ile Leu Phe Le #u Ala His Val Cys Leu   1               5  #                 10  #                 15 Val Asn His Gln Gln Ala Ala Glu Pro Gly Tr #p Lys Gln His Cys Cys              20      #             25      #             30 Asn Trp Glu Gly His Arg Val Leu Xaa          35          #         40 <210> SEQ ID NO 97 <211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (14) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (50) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 97 Met Leu Cys His Val Tyr Leu Leu Leu Val Gl #y His Ala Xaa Phe Ser   1               5  #                 10  #                 15 Val Gly Leu Met Gly Gln Arg Lys Leu Arg Cy #s Ser Ile Asn Ser Ala              20      #             25      #             30 Leu Arg Ser Ala Val Ser Ser Ala Trp Asn Se #r Ser Ile Cys Phe Asn          35          #         40          #         45 Ser Xaa      50 <210> SEQ ID NO 98 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (58) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 98 Met Ser Glu Trp Cys Gln Pro Asp Gln Ile Le #u Leu Gln Phe Pro Val   1               5  #                 10  #                 15 Leu Ala Thr Met Ser Val Ala Phe Leu Ile Gl #n Arg Cys Phe Cys Phe              20      #             25      #             30 Trp Trp Phe Val Leu Asn Ala Phe Ser Ile Pr #o Ser Gly Thr Glu Lys          35          #         40          #         45 Lys Arg Ile Val Phe Lys Lys Trp Leu Xaa      50              #     55 <210> SEQ ID NO 99 <211> LENGTH: 52 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (52) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 99 Met Lys Val Val Val Val Met Val Val Ile Le #u Val Val Val Thr Leu   1               5  #                 10  #                 15 Val Val Val Val Met Val Val Ile Leu Val Me #t Val Val Met Val Val              20      #             25      #             30 Ala Leu Val Thr Leu Thr Trp Gly Pro Val Al #a Val Thr Val Asp Ala          35          #         40          #         45 Gly Ser Trp Xaa      50 <210> SEQ ID NO 100 <211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (45) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 100 Met Pro His Phe Leu Arg Trp Leu Leu Thr Th #r Phe Arg Ile Arg Ala   1               5  #                 10  #                 15 Ser Cys Gly Ser Thr Pro Cys Trp Ser Pro Se #r His Leu Gly Cys Leu              20      #             25      #             30 Gln Pro Ala Leu Pro Arg Asp Leu Ser His Le #u Glu Xaa          35          #         40          #         45 <210> SEQ ID NO 101 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (58) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 101 Met Ser Thr Lys Ile Leu Gln Phe Leu Phe Se #r Ser Cys Cys Trp Val   1               5  #                 10  #                 15 Pro Pro Met Leu Phe Leu Phe Lys Asn Thr Ly #s Cys Arg Thr Ser Leu              20      #             25      #             30 Leu Tyr Cys Phe Tyr Phe Ile Leu Leu Thr Cy #s Ser Leu Ser Glu Tyr          35          #         40          #         45 Asp Ser Leu Leu Ser Ser Lys Val Phe Xaa      50              #     55 <210> SEQ ID NO 102 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (41) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 102 Met Phe Trp Phe Trp Phe Leu Leu Ser Leu Se #r Phe Gln Gln Val Glu   1               5  #                 10  #                 15 Gln Gln Gln Val Phe Gln Cys Ile Cys Cys Th #r Arg Thr Lys Tyr Lys              20      #             25      #             30 Ser Val Trp His Gln Lys Ser Lys Xaa          35          #         40 <210> SEQ ID NO 103 <211> LENGTH: 143 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (104) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (105) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (115) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (143) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 103 Met Thr Leu Ile Glu Val Leu Val Ser Val Le #u Ile Leu Ala Val Gly   1               5  #                 10  #                 15 Leu Leu Arg Ala Ala Val Ile Gln Leu Asn Al #a Leu Lys Tyr Thr Asp              20      #             25      #             30 Ser Ser Arg Met Thr Ser Gln Ala Ser Phe Il #e Ala Tyr Asp Met Leu          35          #         40          #         45 Asp Arg Ile Arg Ala Asn Ser Gly Ala Asp Ty #r Ser Trp Gly Gln Gly      50              #     55              #     60 Glu Arg Ala Pro Ser Thr Thr Ser Val Ala Se #r Val Arg Asp Leu Asp  65                  # 70                  # 75                  # 80 Leu His Asp Phe Glu Ala Asn Ile Val Gly Ph #e Ala Gly Glu Ser Ala                  85  #                 90  #                 95 Lys Gly Ser Val Ala Val Asn Xaa Xaa Glu Va #l Thr Ile Ser Ile Ser             100       #           105       #           110 Trp Asp Xaa Ser Arg Gly Ala Asn Ala Gln Gl #y Thr Arg Glu Thr Phe         115           #       120           #       125 Thr Leu Thr Ser Arg Val Ala Val Asp Pro Ar #g Val Leu Pro Xaa     130               #   135               #   140 <210> SEQ ID NO 104 <211> LENGTH: 44 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (44) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 104 Met Ala Phe Phe Phe Ala Leu Phe Val Ile Ph #e Phe Val Ile Val Val   1               5  #                 10  #                 15 Gln Met Glu Ser His Ser Gly Leu Gly Lys Ly #s Ser Lys Ile Leu Ser              20      #             25      #             30 Gly Gly Gln Gly Glu Glu Val Tyr Phe Leu As #p Xaa          35          #         40 <210> SEQ ID NO 105 <211> LENGTH: 63 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (63) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 105 Met Tyr Phe Tyr Leu Ala Val Lys Pro Pro Le #u Leu Trp Ala Arg Pro   1               5  #                 10  #                 15 Gln Val Ser Cys Arg Leu Ser Val Ser Leu Al #a Trp Ser Tyr His Leu              20      #             25      #             30 His Leu Trp Ala Leu Phe Leu Phe Ser Ile Le #u Leu Gln Cys Arg Ala          35          #         40          #         45 Arg Phe Leu Leu Leu Leu Val Leu Ser Gln Th #r Gln Asp Leu Xaa      50              #     55              #     60 <210> SEQ ID NO 106 <211> LENGTH: 283 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (283) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 106 Met Gly Ser Pro Gly Met Val Leu Gly Leu Le #u Val Gln Ile Trp Ala   1               5  #                 10  #                 15 Leu Gln Glu Ala Ser Ser Leu Ser Val Gln Gl #n Gly Pro Asn Leu Leu              20      #             25      #             30 Gln Val Arg Gln Gly Ser Gln Ala Thr Leu Va #l Cys Gln Val Asp Gln          35          #         40          #         45 Ala Thr Ala Trp Glu Arg Leu Arg Val Lys Tr #p Thr Lys Asp Gly Ala      50              #     55              #     60 Ile Leu Cys Gln Pro Tyr Ile Thr Asn Gly Se #r Leu Ser Leu Gly Val  65                  # 70                  # 75                  # 80 Cys Gly Pro Gln Gly Arg Leu Ser Trp Gln Al #a Pro Ser His Leu Thr                  85  #                 90  #                 95 Leu Gln Leu Asp Pro Val Ser Leu Asn His Se #r Gly Ala Tyr Val Cys             100       #           105       #           110 Trp Ala Ala Val Glu Ile Pro Glu Leu Glu Gl #u Ala Glu Gly Asn Ile         115           #       120           #       125 Thr Arg Leu Phe Val Asp Pro Asp Asp Pro Th #r Gln Asn Arg Asn Arg     130               #   135               #   140 Ile Ala Ser Phe Pro Gly Phe Leu Phe Val Le #u Leu Gly Val Gly Ser 145                 1 #50                 1 #55                 1 #60 Met Gly Val Ala Ala Ile Val Trp Gly Ala Tr #p Phe Trp Gly Arg Arg                 165   #               170   #               175 Ser Cys Gln Gln Arg Asp Ser Gly Asn Ser Pr #o Gly Asn Ala Phe Tyr             180       #           185       #           190 Ser Asn Val Leu Tyr Arg Pro Arg Gly Ala Pr #o Lys Lys Ser Glu Asp         195           #       200           #       205 Cys Ser Gly Glu Gly Lys Asp Gln Arg Gly Gl #n Ser Ile Tyr Ser Thr     210               #   215               #   220 Ser Phe Pro Gln Pro Ala Pro Arg Gln Pro Hi #s Leu Ala Ser Arg Pro 225                 2 #30                 2 #35                 2 #40 Cys Pro Ser Pro Arg Pro Cys Pro Ser Pro Ar #g Pro Gly His Pro Val                 245   #               250   #               255 Ser Met Val Arg Val Ser Pro Arg Pro Ser Pr #o Thr Gln Gln Pro Arg             260       #           265       #           270 Pro Lys Gly Phe Pro Lys Val Gly Glu Glu Xa #a         275           #       280 <210> SEQ ID NO 107 <211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (98) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 107 Met Cys Lys Leu Cys Phe Tyr Leu Tyr Leu Cy #s Thr Trp Phe Pro Phe   1               5  #                 10  #                 15 Gly Ala Ser Gly Leu Phe Trp Asp Lys Trp Cy #s Leu Pro Arg His Leu              20      #             25      #             30 Pro Val Val Ser Gly Gln Glu Gln Leu Ser Se #r Ser Leu Pro Ala Ala          35          #         40          #         45 Leu Leu Phe Leu Gly Arg Arg Trp Arg Pro Pr #o Leu Arg Val Ser Pro      50              #     55              #     60 Gly Leu Ser Phe Arg Gly Gly Arg Ala Gly Gl #u Pro Gln Gly Trp Gly  65                  # 70                  # 75                  # 80 Asp Ser Trp Glu Met Glu Val Ala Pro Ala Pr #o Leu Asp Gln Tyr Trp                  85  #                 90  #                 95 Leu Xaa <210> SEQ ID NO 108 <211> LENGTH: 62 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (62) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 108 Met Cys Leu Leu Leu Leu Trp Leu Thr Thr Ph #e Gln Arg Thr Ser Gly   1               5  #                 10  #                 15 Ala Leu Arg Arg Gly Gly Leu Ser Ser Pro Al #a Trp Ala Met Arg Ser              20      #             25      #             30 Pro Ser Val Tyr Ser Thr Gln Thr Pro Ser Pr #o Met Met Ser Thr Gly          35          #         40          #         45 Thr Leu Arg Gly Leu Ser Gly Ala Met Cys As #n Leu Ser Xaa      50              #     55              #     60 <210> SEQ ID NO 109 <211> LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (47) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 109 Met Lys Leu Cys Lys Leu Thr Gln Cys Ser Ph #e Leu Leu Lys Ser Leu   1               5  #                 10  #                 15 Ile Leu Leu Leu Glu Gln Leu Asn Val Ser Me #t Gly Phe Val Ala Ala              20      #             25      #             30 Phe Asp Val Leu Val Gly Cys Ser Ile Cys Ph #e Glu Lys His Xaa          35          #         40          #         45 <210> SEQ ID NO 110 <211> LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (47) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 110 Met Thr Thr Phe Ser Leu Cys Ser Gln Leu Al #a Leu Leu Cys Ala Cys   1               5  #                 10  #                 15 Thr Ser Leu Val Ser Leu Pro Pro Phe Val As #p Tyr Lys Asp Thr Ser              20      #             25      #             30 Pro Val Gly Pro Glu Pro His Cys Lys Gly Le #u Ile Leu Thr Xaa          35          #         40          #         45 <210> SEQ ID NO 111 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (32) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (33) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (41) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (42) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 111 Met Asn Ile Leu Val Cys Val Phe Trp Leu Tr #p Gly Gly Val Ala Gly   1               5  #                 10  #                 15 Ser Trp Gly Arg His Ile Phe Ile Phe Thr Se #r Val Lys Asn Val Xaa              20      #             25      #             30 Xaa Ala Ser His Cys Ala Trp Pro Xaa Xaa          35          #         40 <210> SEQ ID NO 112 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 112 Met Gly Gly Ile Ala Leu Pro Ser Leu Ser Le #u Cys Leu Leu Ser Ala   1               5  #                 10  #                 15 Gly Ser His Cys Ile Ser Pro Ala Asp Gln Gl #u Thr Gly Pro Lys Val              20      #             25      #             30 Thr Ala Pro Gln Gly Asn Phe Leu Pro          35          #         40 <210> SEQ ID NO 113 <211> LENGTH: 44 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (44) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 113 Met Ile Val Leu Lys Trp Ile Phe Leu Ala Cy #s Val His Glu Cys Met   1               5  #                 10  #                 15 Cys Lys Pro Leu Lys Cys Phe Leu Glu Lys Il #e Leu Glu Val Leu Ile              20      #             25      #             30 Met Val Lys Leu Lys Met Gly Val Leu Pro Al #a Xaa          35          #         40 <210> SEQ ID NO 114 <211> LENGTH: 182 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 114 Met Glu Pro Gln Leu Gly Pro Glu Ala Ala Al #a Leu Arg Pro Gly Trp   1               5  #                 10  #                 15 Leu Ala Leu Leu Leu Trp Val Ser Ala Leu Se #r Cys Ser Phe Ser Leu              20      #             25      #             30 Pro Ala Ser Ser Leu Ser Ser Leu Val Pro Gl #n Val Arg Thr Ser Tyr          35          #         40          #         45 Asn Phe Gly Arg Thr Phe Leu Gly Leu Asp Ly #s Cys Asn Ala Cys Ile      50              #     55              #     60 Gly Thr Ser Ile Cys Lys Lys Phe Phe Lys Gl #u Glu Ile Arg Ser Asp  65                  # 70                  # 75                  # 80 Asn Trp Leu Ala Ser His Leu Gly Leu Pro Pr #o Asp Ser Leu Leu Ser                  85  #                 90  #                 95 Tyr Pro Ala Asn Tyr Ser Asp Asp Ser Lys Il #e Trp Arg Pro Val Glu             100       #           105       #           110 Ile Phe Arg Leu Val Ser Lys Tyr Gln Asn Gl #u Ile Ser Asp Arg Lys         115           #       120           #       125 Ile Cys Ala Ser Ala Ser Ala Pro Lys Thr Cy #s Ser Ile Glu Arg Val     130               #   135               #   140 Leu Arg Lys Thr Glu Arg Phe Gln Lys Trp Le #u Gln Ala Lys Arg Leu 145                 1 #50                 1 #55                 1 #60 Thr Pro Asp Leu Val Gln Asp Cys His Gln Gl #y Gln Arg Glu Leu Lys                 165   #               170   #               175 Phe Leu Cys Met Leu Arg             180 <210> SEQ ID NO 115 <211> LENGTH: 81 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (81) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 115 Met Ala Leu Gly Ser Met Tyr Leu Val Leu Th #r Leu Ile Val Ala Lys   1               5  #                 10  #                 15 Val Leu Arg Gly Ala Glu Pro Cys Cys Gly Pr #o Leu Lys Asn Arg Val              20      #             25      #             30 Leu Arg Pro Cys Pro Leu Pro Val His Cys Pr #o Leu Pro Ile Pro Ser          35          #         40          #         45 Pro Ala Glu Gly Ile Pro Trp Val Ala Tyr Le #u Pro Ile Arg Trp Phe      50              #     55              #     60 Ile Ser Cys Cys Pro Gly His Cys Ile Gln Il #e Pro Met Cys Thr Ser  65                  # 70                  # 75                  # 80 Xaa <210> SEQ ID NO 116 <211> LENGTH: 49 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (49) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 116 Met Ser Cys Glu Asn Asn Leu Lys Lys Lys As #n Thr Thr Leu Leu Ser   1               5  #                 10  #                 15 Tyr Leu Ile Phe Leu Ala Leu Val Met Tyr Le #u Thr Phe Met Phe Leu              20      #             25      #             30 Ser Ser Val Ser Thr Ser Arg Ile Ser Leu Se #r Asn Ser Met Ile Ile          35          #         40          #         45 Xaa <210> SEQ ID NO 117 <211> LENGTH: 204 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (31) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (93) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (99) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (115) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (151) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (204) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 117 Met Val Gly Leu Met His Ile Gly Phe Gly Il #e Val Leu Cys Leu Ile   1               5  #                 10  #                 15 Ser Phe Ser Phe Arg Glu Val Leu Gly Phe Al #a Ser Thr Ala Xaa Ile              20      #             25      #             30 Gly Gly Tyr Pro Phe Trp Gly Gly Leu Ser Ph #e Ile Ile Ser Gly Ser          35          #         40          #         45 Leu Ser Val Ser Ala Ser Lys Glu Leu Ser Ar #g Cys Leu Val Lys Gly      50              #     55              #     60 Ser Leu Gly Met Asn Ile Gly Arg Ser Ile Le #u Ala Phe Ile Gly Val  65                  # 70                  # 75                  # 80 Ile Leu Leu Leu Val Asp Met Cys Ile Asn Gl #y Val Xaa Gly Gln Asp                  85  #                 90  #                 95 Tyr Trp Xaa Val Leu Ser Gly Lys Gly Ile Se #r Ala Thr Leu Met Ile             100       #           105       #           110 Phe Ser Xaa Leu Glu Phe Phe Val Ala Cys Al #a Thr Ala His Phe Ala         115           #       120           #       125 Asn Gln Ala Asn Thr Thr Thr Asn Met Ser Va #l Leu Val Ile Pro Asn     130               #   135               #   140 Met Tyr Glu Ser Asn Pro Xaa Thr Pro Ala Se #r Ser Ser Ala Pro Pro 145                 1 #50                 1 #55                 1 #60 Arg Cys Asn Asn Tyr Ser Ala Asn Ala Pro Ly #s Arg Lys Arg Gly Ile                 165   #               170   #               175 Ser Leu Ile Ser Trp Arg Lys Thr Thr Cys Ly #s Asn Phe Leu Arg Arg             180       #           185       #           190 Cys Leu Leu Leu Ser Thr Met Ile Ser Ser Le #u Xaa         195           #       200 <210> SEQ ID NO 118 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (19) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 118 Ser Leu Asp Ala Phe Arg Leu Ile Arg Ala Me #t Gly Ala Thr Gly Leu   1               5  #                 10  #                 15 Ser Phe Xaa <210> SEQ ID NO 119 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (13) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 119 Leu Val Leu Trp Ile Val Met Leu Thr Tyr Al #a Thr Xaa   1               5  #                 10 <210> SEQ ID NO 120 <211> LENGTH: 80 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (80) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 120 Met Glu Pro Gln Leu Gly Pro Glu Ala Ala Al #a Leu Arg Pro Gly Trp   1               5  #                 10  #                 15 Leu Ala Leu Leu Leu Trp Val Ser Ala Leu Se #r Cys Ser Phe Ser Leu              20      #             25      #             30 Pro Ala Ser Ser Leu Ser Ser Leu Val Pro Gl #n Val Arg Thr Ser Tyr          35          #         40          #         45 Asn Phe Gly Arg Thr Phe Leu Gly Leu Asp Ly #s Cys Asn Ala Cys Ile      50              #     55              #     60 Gly Thr Ser Ile Cys Lys Lys Phe Phe Lys Gl #u Arg Asn Lys Ile Xaa  65                  # 70                  # 75                  # 80 <210> SEQ ID NO 121 <211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (96) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (107) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (111) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (115) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (122) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (132) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 121 Met Glu Pro Gln Leu Gly Pro Glu Ala Ala Al #a Leu Arg Pro Gly Trp   1               5  #                 10  #                 15 Leu Ala Leu Leu Leu Trp Val Ser Ala Leu Se #r Cys Ser Phe Ser Leu              20      #             25      #             30 Pro Ala Ser Ser Leu Ser Ser Leu Val Pro Gl #n Val Arg Thr Ser Tyr          35          #         40          #         45 Asn Phe Gly Arg Thr Phe Leu Gly Leu Asp Ly #s Cys Asn Ala Cys Ile      50              #     55              #     60 Gly Thr Ser Ile Cys Lys Lys Phe Phe Lys Gl #u Glu Ile Arg Ser Asp  65                  # 70                  # 75                  # 80 Asn Trp Leu Ala Ser His Leu Gly Thr Ala Se #r Arg Phe Pro Leu Xaa                  85  #                 90  #                 95 Ser Tyr Pro Cys Lys Leu Leu Gln Met Ile Xa #a Lys Ile Trp Xaa Pro             100       #           105       #           110 Cys Gly Xaa Leu Leu Thr Gly Gln Gln Xaa Se #r Asn Glu Ile Ser Lys         115           #       120           #       125 Gln Glu Ile Xaa Cys Leu Leu His Pro Pro Pr #o Lys Asn Leu His Ile     130               #   135               #   140 Asp Val 145 <210> SEQ ID NO 122 <211> LENGTH: 81 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (81) <223> OTHER INFORMATION: Xaa equals stop translati #on <400> SEQUENCE: 122 Met Ala Leu Gly Ser Met Tyr Leu Val Leu Th #r Leu Ile Val Ala Lys   1               5  #                 10  #                 15 Val Leu Arg Gly Ala Glu Pro Cys Cys Gly Pr #o Leu Lys Asn Arg Val              20      #             25      #             30 Leu Arg Pro Cys Pro Leu Pro Val His Cys Pr #o Leu Pro Ile Pro Ser          35          #         40          #         45 Pro Ala Glu Gly Ile Pro Trp Val Ala Tyr Le #u Pro Ile Arg Trp Phe      50              #     55              #     60 Ile Ser Cys Cys Pro Gly His Cys Ile Gln Il #e Pro Met Cys Thr Ser  65                  # 70                  # 75                  # 80 Xaa <210> SEQ ID NO 123 <211> LENGTH: 337 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 123 Glu Pro His Arg Gly Pro His Leu Pro Pro As #p Leu Gly His His His   1               5  #                 10  #                 15 Gly Gln Arg Pro Gly Leu Gln Asn Ile Asn Va #l Phe Leu Arg Asn Thr              20      #             25      #             30 Val Lys Val Thr Gly Val Val Val Phe Met Ph #e Ser Leu Ser Trp Gln          35          #         40          #         45 Leu Ser Leu Val Thr Phe Met Gly Phe Pro Il #e Ile Met Met Val Ser      50              #     55              #     60 Asn Ile Tyr Gly Lys Tyr Tyr Lys Arg Leu Se #r Lys Glu Val Gln Asn  65                  # 70                  # 75                  # 80 Ala Leu Ala Arg Ala Ser Asn Thr Ala Glu Gl #u Thr Ile Ser Ala Met                  85  #                 90  #                 95 Lys Thr Val Arg Ser Phe Ala Asn Glu Glu Gl #u Glu Ala Glu Val Tyr             100       #           105       #           110 Leu Arg Lys Leu Gln Gln Val Tyr Lys Leu As #n Arg Lys Glu Ala Ala         115           #       120           #       125 Ala Tyr Met Tyr Tyr Val Trp Gly Ser Gly Le #u Thr Leu Leu Val Val     130               #   135               #   140 Gln Val Ser Ile Leu Tyr Tyr Gly Gly His Le #u Val Ile Ser Gly Gln 145                 1 #50                 1 #55                 1 #60 Met Thr Ser Gly Asn Leu Ile Ala Phe Ile Il #e Tyr Glu Phe Val Leu                 165   #               170   #               175 Gly Asp Cys Met Glu Asn Val Ser Phe Ser Le #u Ser Pro Gly Lys Val             180       #           185       #           190 Thr Ala Leu Val Gly Pro Ser Gly Ser Gly Ly #s Ser Ser Cys Val Asn         195           #       200           #       205 Ile Leu Glu Asn Phe Tyr Pro Leu Glu Gly Gl #y Arg Val Leu Leu Asp     210               #   215               #   220 Gly Lys Pro Ile Ser Ala Tyr Asp His Lys Ty #r Leu His Arg Val Ile 225                 2 #30                 2 #35                 2 #40 Ser Leu Val Ser Gln Glu Pro Val Leu Phe Al #a Arg Ser Ile Thr Asp                 245   #               250   #               255 Asn Ile Ser Tyr Gly Leu Pro Thr Val Pro Ph #e Glu Met Val Val Glu             260       #           265       #           270 Ala Ala Gln Lys Ala Asn Ala His Gly Phe Il #e Met Glu Leu Gln Asp         275           #       280           #       285 Gly Tyr Ser Thr Glu Thr Gly Glu Lys Gly Al #a Gln Leu Ser Gly Gly     290               #   295               #   300 Gln Lys Gln Arg Val Ala Trp Pro Gly Leu Tr #p Cys Gly Thr Pro Gln 305                 3 #10                 3 #15                 3 #20 Ser Ser Ser Trp Met Lys Pro Pro Ala Leu Tr #p Met Pro Arg Ala Ser                 325   #               330   #               335 Ile <210> SEQ ID NO 124 <211> LENGTH: 315 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 124 Met Ser Ser Ala Thr Trp Thr Ala Ala Ser Tr #p Arg Thr Ser Ala Thr   1               5  #                 10  #                 15 Ser Thr Ser Leu Thr Arg Cys Trp Ile Ser Gl #y Gln Pro Ala Cys Thr              20      #             25      #             30 Ala Ala Ala Cys Cys Trp Gly Ala Thr Ile Gl #y Val Ala Lys Asn Ser          35          #         40          #         45 Ala Leu Gly Pro Arg Arg Leu Arg Ala Ser Tr #p Leu Val Ile Thr Leu      50              #     55              #     60 Val Cys Leu Phe Val Gly Ile Tyr Ala Met Va #l Lys Leu Leu Leu Phe  65                  # 70                  # 75                  # 80 Ser Glu Val Arg Arg Pro Ile Arg Asp Pro Tr #p Phe Trp Ala Leu Phe                  85  #                 90  #                 95 Val Trp Thr Tyr Ile Ser Leu Gly Ala Ser Ph #e Leu Leu Trp Trp Leu             100       #           105       #           110 Leu Ser Thr Val Arg Pro Gly Thr Gln Ala Le #u Glu Pro Gly Ala Ala         115           #       120           #       125 Thr Glu Ala Glu Gly Phe Pro Gly Ser Gly Ar #g Pro Pro Pro Glu Gln     130               #   135               #   140 Ala Ser Gly Ala Thr Leu Gln Lys Leu Leu Se #r Tyr Thr Lys Pro Asp 145                 1 #50                 1 #55                 1 #60 Val Ala Phe Leu Val Ala Ala Ser Phe Phe Le #u Ile Val Ala Ala Leu                 165   #               170   #               175 Gly Glu Thr Phe Leu Pro Tyr Tyr Thr Gly Ar #g Ala Ile Asp Gly Ile             180       #           185       #           190 Val Ile Gln Lys Ser Met Asp Gln Phe Ser Th #r Ala Val Val Ile Val         195           #       200           #       205 Cys Leu Leu Ala Ile Gly Ser Ser Phe Ala Al #a Gly Ile Arg Gly Gly     210               #   215               #   220 Ile Phe Thr Leu Ile Phe Ala Arg Leu Asn Il #e Arg Leu Arg Asn Cys 225                 2 #30                 2 #35                 2 #40 Leu Phe Arg Ser Leu Val Ser Gln Glu Thr Se #r Phe Phe Asp Glu Asn                 245   #               250   #               255 Arg Thr Gly Asp Leu Ile Ser Arg Leu Thr Se #r Asp Thr Thr Met Val             260       #           265       #           270 Ser Asp Leu Val Ser Arg Thr Ser Met Ser Se #r Cys Gly Thr Gln Ser         275           #       280           #       285 Arg Ser Arg Ala Trp Trp Ser Ser Cys Ser Al #a Ser His Gly Ser Ser     290               #   295               #   300 Pro Trp Ser Pro Ser Trp Ala Ser Pro Ser Se #r 305                 3 #10                 3 #15 <210> SEQ ID NO 125 <211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 125 His Leu Leu Arg Pro Ala His Cys Ala Phe Ar #g Asp Gly Gly Gly Gly   1               5  #                 10  #                 15 Arg Thr Glu Gly Gln Cys Pro Arg Leu His Hi #s Gly Thr Pro Gly Arg              20      #             25      #             30 Leu Gln His Arg Asp Arg Gly Glu Gly Arg Pr #o Ala Val Arg Trp Pro          35          #         40          #         45 Glu Ala Ala Gly Gly Met Ala Arg Ala Leu Va #l Arg Asn Pro Pro Val      50              #     55              #     60 Leu Ile Leu Asp Glu Ala Thr Ser Ala Leu As #p Ala Glu Ser Glu Tyr  65                  # 70                  # 75                  # 80 Leu Ile Gln Gln Ala Ile His Gly Asn Leu Gl #n Lys His Thr Val Leu                  85  #                 90  #                 95 Ile Ile Ala His Arg Leu Ser Thr Val Glu Hi #s Ala His Leu Ile Val             100       #           105       #           110 Val Leu Asp Lys Gly Arg Val Val Gln Gln Gl #y Thr His Gln Gln Leu         115           #       120           #       125 Leu Ala Gln Gly Gly Leu Tyr Ala Lys Leu Va #l Gln Arg Gln Met Leu     130               #   135               #   140 Gly Leu Gln Pro Ala Ala Asp Phe Thr Ala Gl #y His Asn Glu Pro Val 145                 1 #50                 1 #55                 1 #60 Ala Asn Gly Ser His Lys Ala                 165 <210> SEQ ID NO 126 <211> LENGTH: 227 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (71) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 126 Arg Leu Thr Lys Thr Ile Ser Phe Ser Leu Gl #n Asn Gln Thr Ala Phe   1               5  #                 10  #                 15 Ile Asn Ser Leu Ala Lys Thr Pro Tyr Gln Al #a Leu Thr Gly Ala Ala              20      #             25      #             30 Leu Ala Gly Ser Tyr Pro Ile Trp Glu Asn Gl #u Asn Thr Leu Ser Trp          35          #         40          #         45 Tyr Leu Pro Ser Pro Thr Thr Leu Leu Ser Pr #o Pro Val Leu Phe Cys      50              #     55              #     60 Val Ile Gln Leu Ile Phe Xaa Leu Pro Ala As #n Trp Ser Gly Thr Cys  65                  # 70                  # 75                  # 80 Thr Leu Val Phe Gln Ala Pro Thr Ile Asn Il #e Leu Pro Pro Asn Gln                  85  #                 90  #                 95 Thr Ile Leu Ile Ser Val Glu Ala Ser Ile Se #r Ser Ser Pro Ile Arg             100       #           105       #           110 Asn Lys Trp Ala Leu His Leu Ile Thr Leu Le #u Thr Gly Leu Gly Ile         115           #       120           #       125 Thr Ala Ala Leu Gly Thr Gly Ile Ala Gly Il #e Thr Thr Ser Ile Thr     130               #   135               #   140 Ser Tyr Gln Thr Leu Phe Thr Thr Leu Ser As #n Thr Val Glu Asp Met 145                 1 #50                 1 #55                 1 #60 His Thr Ser Ile Thr Ser Leu Gln Arg Gln Le #u Asp Phe Leu Val Gly                 165   #               170   #               175 Val Ile Leu Gln Asn Trp Arg Val Leu Asp Le #u Leu Thr Thr Glu Lys             180       #           185       #           190 Gly Gly Thr Cys Ile Tyr Leu Gln Glu Glu Cy #s Cys Phe Cys Val Asn         195           #       200           #       205 Glu Ser Gly Ile Val His Ile Ala Val Arg Ar #g Leu His Asp Arg Ala     210               #   215               #   220 Ala Glu Leu 225 <210> SEQ ID NO 127 <211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 127 Tyr Pro Ile Trp Glu Asn Glu Asn Thr Leu Se #r Trp Tyr Leu Pro Ser   1               5  #                 10  #                 15 Pro Thr Thr Leu Leu Ser Pro Pro Val Leu Ph #e Cys Val              20      #             25 <210> SEQ ID NO 128 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 128 Arg Val Leu Asp Leu Leu Thr Thr Glu Lys Gl #y Gly Thr Cys Ile Tyr   1               5  #                 10  #                 15 Leu Gln Glu Glu Cys Cys Phe Cys Val Asn Gl #u              20      #             25 <210> SEQ ID NO 129 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 129 Phe Ser Leu Gly Arg Arg His Cys Leu Gly   1               5  #                 10 <210> SEQ ID NO 130 <211> LENGTH: 123 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (64) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (83) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 130 Glu His Pro Thr Ala Asp Arg Ala Gly Cys Se #r Ala Ser Gly Ala Cys   1               5  #                 10  #                 15 Tyr Ser Leu His His Ala Thr Met Lys Arg Gl #n Ala Ala Glu Glu Ala              20      #             25      #             30 Cys Ile Leu Arg Gly Gly Ala Leu Ser Thr Va #l Arg Ala Gly Ala Glu          35          #         40          #         45 Leu Arg Ala Val Leu Ala Leu Leu Arg Ala Gl #y Pro Gly Pro Gly Xaa      50              #     55              #     60 Gly Ser Lys Asp Leu Leu Phe Trp Val Ala Le #u Glu Arg Arg Arg Ser  65                  # 70                  # 75                  # 80 His Cys Xaa Leu Glu Asn Glu Pro Leu Arg Gl #y Phe Ser Trp Leu Ser                  85  #                 90  #                 95 Ser Asp Pro Gly Gly Leu Glu Ser Asp Thr Le #u Gln Trp Val Glu Glu             100       #           105       #           110 Pro Gln Arg Ser Cys Thr Ala Arg Arg Trp Va #l         115           #       120 <210> SEQ ID NO 131 <211> LENGTH: 344 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (19) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 131 Ser Arg Pro Pro Val Gly Ser Ser Pro Gln Le #u Glu Gly Asp Ala Met   1               5  #                 10  #                 15 Pro Pro Xaa Arg Gln Arg Tyr Leu Cys Lys Ty #r Gln Phe Glu Val Leu              20      #             25      #             30 Cys Pro Ala Pro Arg Pro Gly Ala Ala Ser As #n Leu Ser Tyr Arg Ala          35          #         40          #         45 Pro Phe Gln Leu His Ser Ala Ala Leu Asp Ph #e Ser Pro Pro Gly Thr      50              #     55              #     60 Glu Val Ser Ala Leu Cys Arg Gly Gln Leu Pr #o Ile Ser Val Thr Cys  65                  # 70                  # 75                  # 80 Ile Ala Asp Glu Ile Gly Ala Arg Trp Asp Ly #s Leu Ser Gly Asp Val                  85  #                 90  #                 95 Leu Cys Pro Cys Pro Gly Arg Tyr Leu Arg Al #a Gly Lys Cys Ala Glu             100       #           105       #           110 Leu Pro Asn Cys Leu Asp Asp Leu Gly Gly Ph #e Ala Cys Glu Cys Ala         115           #       120           #       125 Thr Gly Phe Glu Leu Gly Lys Asp Gly Arg Se #r Cys Val Thr Ser Gly     130               #   135               #   140 Glu Gly Gln Pro Thr Leu Gly Gly Thr Gly Va #l Pro Thr Arg Arg Pro 145                 1 #50                 1 #55                 1 #60 Pro Ala Thr Ala Thr Ser Pro Val Pro Gln Ar #g Thr Trp Pro Ile Arg                 165   #               170   #               175 Val Asp Glu Lys Leu Gly Glu Thr Pro Leu Va #l Pro Glu Gln Asp Asn             180       #           185       #           190 Ser Val Thr Ser Ile Pro Glu Ile Pro Arg Tr #p Gly Ser Gln Ser Thr         195           #       200           #       205 Met Ser Thr Leu Gln Met Ser Leu Gln Ala Gl #u Ser Lys Ala Thr Ile     210               #   215               #   220 Thr Pro Ser Gly Ser Val Ile Ser Lys Phe As #n Ser Thr Thr Ser Ser 225                 2 #30                 2 #35                 2 #40 Ala Thr Pro Gln Ala Phe Asp Ser Ser Ser Al #a Val Val Phe Ile Phe                 245   #               250   #               255 Val Ser Thr Ala Val Val Val Leu Val Ile Le #u Thr Met Thr Val Leu             260       #           265       #           270 Gly Leu Val Lys Leu Cys Phe His Glu Ser Pr #o Ser Ser Gln Pro Arg         275           #       280           #       285 Lys Glu Ser Met Gly Pro Pro Gly Trp Arg Va #l Ile Leu Lys Pro Ala     290               #   295               #   300 Ala Leu Gly Ser Ser Ser Ala His Cys Thr As #n Asn Gly Val Lys Val 305                 3 #10                 3 #15                 3 #20 Gly Asp Cys Asp Leu Arg Asp Arg Ala Glu Gl #y Ala Leu Leu Ala Glu                 325   #               330   #               335 Ser Pro Leu Gly Ser Ser Asp Ala             340 <210> SEQ ID NO 132 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 132 Arg Tyr Leu Thr Leu Thr His   1               5 <210> SEQ ID NO 133 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 133 Cys Asn Thr Pro Trp Ala   1               5 <210> SEQ ID NO 134 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 134 Ala Pro Val Ile Phe Ser His Ser   1               5 <210> SEQ ID NO 135 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 135 Arg Asn Val Pro Asp Asp   1               5 <210> SEQ ID NO 136 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 136 Gly Leu Glu Asp Val Ser   1               5 <210> SEQ ID NO 137 <211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 137 Val Glu Gly Gly His Ser Leu Asp Asn Ser Le #u Ser Ile Leu Arg Thr   1               5  #                 10  #                 15 Phe Tyr Met Leu Gly Val Arg              20 <210> SEQ ID NO 138 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 138 Val Glu Gly Gly His Ser   1               5 <210> SEQ ID NO 139 <211> LENGTH: 190 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 139 Thr Trp Leu Arg Leu Gly Ser Ser Gln Ile Tr #p Leu Gly Thr Ala Pro   1               5  #                 10  #                 15 Arg Gly Pro Arg Ile His Pro Glu Gln Ala Gl #y Leu Ala Gly Ala Pro              20      #             25      #             30 Val Lys Ser Thr Ser Ser Glu Glu Ser Gln Pr #o Gly Gly Gln Cys Gln          35          #         40          #         45 Ser Ser Gly Gly Ala Gln Thr Leu Pro Ser Le #u Arg Ala Ala Pro Val      50              #     55              #     60 Ala Ala Leu Gly Ser Leu Ser Ser Tyr Pro As #p Ser Cys Pro Arg Ala  65                  # 70                  # 75                  # 80 Thr Thr Pro Glu Leu Cys Pro Gly Ala Pro Th #r Leu His Leu Ala Asp                  85  #                 90  #                 95 Ser Ile Ser Gly Pro Val Ser Pro Pro Gly Se #r Ser Leu Gly Pro Asp             100       #           105       #           110 Ala Trp Thr Leu Cys Ala Lys His His Gln Al #a Lys Gly Met Thr Leu         115           #       120           #       125 Gly Thr Pro Lys Val Leu Arg Leu Gln Pro Va #l Ser Pro Cys Trp Gly     130               #   135               #   140 Pro Lys Ser Trp Arg Val Pro Gly Pro Phe Gl #n Pro Gly Arg Arg Arg 145                 1 #50                 1 #55                 1 #60 Gly Glu Ser Arg Gln Gln Gly Arg Gly Lys Ar #g Arg Ser Ala Arg Ser                 165   #               170   #               175 Ala Gln Ser Pro Thr Gly Pro Glu Ser Ala Al #a Trp Pro Cys             180       #           185       #           190 <210> SEQ ID NO 140 <211> LENGTH: 129 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 140 Thr Val Ala Thr Ala Cys Val Trp Ala Ala Cy #s Thr Gly Cys Trp Ala   1               5  #                 10  #                 15 Arg Pro Pro Val Pro Thr Trp Ala Gly Cys Al #a Ala Arg Cys Ala Ala              20      #             25      #             30 Glu Asp Ala Arg Ala Gly Val Gly Asp Leu Pr #o Ala Thr Gly Gly Ala          35          #         40          #         45 Ala Thr Gly Arg Arg Ala Leu Thr Pro Ala Pr #o Pro Arg Gly Pro Cys      50              #     55              #     60 Ile Leu Ser Pro Gln Pro Trp Ala Leu Gly Le #u Pro Gly Ala Pro Leu  65                  # 70                  # 75                  # 80 Pro Ala Ala Leu Pro Gly Arg Ala Arg Gly Ar #g Pro Gly Leu Pro Ala                  85  #                 90  #                 95 Leu Pro Ala Leu Ser Thr Leu Pro Gly Cys Pr #o Ala Leu Asp Pro Ala             100       #           105       #           110 Gly Ala Gly Thr Leu Cys Pro Pro Pro Gly Al #a Ala Glu Pro Ala Gly         115           #       120           #       125 Pro <210> SEQ ID NO 141 <211> LENGTH: 90 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 141 Arg Ser Gly Gln Pro Gly Glu Gly Ser Met Le #u Arg Lys Phe Ser Leu   1               5  #                 10  #                 15 Gln Arg Leu Leu Ser Pro Leu Asp Gln Ala Gl #n Thr Arg Trp Gly Leu              20      #             25      #             30 Ala Leu Ala Cys Val Ala Gly Asp Lys Gly Pr #o Pro Arg Pro Trp Asn          35          #         40          #         45 Ile Ser Ser Ala Pro Ala His Pro His Val Th #r Thr Pro Gly Met Glu      50              #     55              #     60 Thr Ser Gly Gly Pro Ala Arg Asp Gly Gly Le #u Ile Leu Glu Arg Glu  65                  # 70                  # 75                  # 80 Ala Ala Phe Asn Lys Pro Ala Pro Gly Glu                  85  #                 90 <210> SEQ ID NO 142 <211> LENGTH: 307 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (135) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (197) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (203) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (219) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (255) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 142 Arg Cys Gln Arg Asn Lys Asp Ile Met Met Se #r Ser Lys Pro Thr Ser   1               5  #                 10  #                 15 His Ala Glu Val Asn Glu Thr Ile Pro Asn Pr #o Tyr Pro Pro Ser Ser              20      #             25      #             30 Phe Met Ala Pro Gly Phe Gln Gln Pro Leu Gl #y Ser Ile Asn Leu Glu          35          #         40          #         45 Asn Gln Ala Gln Gly Ala Gln Arg Ala Gln Pr #o Tyr Gly Ile Thr Ser      50              #     55              #     60 Pro Gly Ile Phe Ala Ser Ser Gln Pro Gly Gl #n Gly Asn Ile Gln Met  65                  # 70                  # 75                  # 80 Ile Asn Pro Ser Val Gly Thr Ala Val Met As #n Phe Lys Glu Glu Ala                  85  #                 90  #                 95 Lys Ala Leu Gly Val Ile Gln Ile Met Val Gl #y Leu Met His Ile Gly             100       #           105       #           110 Phe Gly Ile Val Leu Cys Leu Ile Ser Phe Se #r Phe Arg Glu Val Leu         115           #       120           #       125 Gly Phe Ala Ser Thr Ala Xaa Ile Gly Gly Ty #r Pro Phe Trp Gly Gly     130               #   135               #   140 Leu Ser Phe Ile Ile Ser Gly Ser Leu Ser Va #l Ser Ala Ser Lys Glu 145                 1 #50                 1 #55                 1 #60 Leu Ser Arg Cys Leu Val Lys Gly Ser Leu Gl #y Met Asn Ile Gly Arg                 165   #               170   #               175 Ser Ile Leu Ala Phe Ile Gly Val Ile Leu Le #u Leu Val Asp Met Cys             180       #           185       #           190 Ile Asn Gly Val Xaa Gly Gln Asp Tyr Trp Xa #a Val Leu Ser Gly Lys         195           #       200           #       205 Gly Ile Ser Ala Thr Leu Met Ile Phe Ser Xa #a Leu Glu Phe Phe Val     210               #   215               #   220 Ala Cys Ala Thr Ala His Phe Ala Asn Gln Al #a Asn Thr Thr Thr Asn 225                 2 #30                 2 #35                 2 #40 Met Ser Val Leu Val Ile Pro Asn Met Tyr Gl #u Ser Asn Pro Xaa Thr                 245   #               250   #               255 Pro Ala Ser Ser Ser Ala Pro Pro Arg Cys As #n Asn Tyr Ser Ala Asn             260       #           265       #           270 Ala Pro Lys Arg Lys Arg Gly Ile Ser Leu Il #e Ser Trp Arg Lys Thr         275           #       280           #       285 Thr Cys Lys Asn Phe Leu Arg Arg Cys Leu Le #u Leu Ser Thr Met Ile     290               #   295               #   300 Ser Ser Leu 305 <210> SEQ ID NO 143 <211> LENGTH: 246 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 143 Met Gly Arg Leu Asp Gly Lys Val Ile Ile Le #u Thr Ala Ala Ala Gln   1               5  #                 10  #                 15 Gly Ile Gly Gln Ala Ala Ala Leu Ala Phe Al #a Arg Glu Gly Ala Lys              20      #             25      #             30 Val Ile Ala Thr Asp Ile Asn Glu Ser Lys Le #u Gln Glu Leu Glu Lys          35          #         40          #         45 Tyr Pro Gly Ile Gln Thr Arg Val Leu Asp Va #l Thr Lys Lys Lys Gln      50              #     55              #     60 Ile Asp Gln Phe Ala Asn Glu Val Glu Arg Le #u Asp Val Leu Phe Asn  65                  # 70                  # 75                  # 80 Val Ala Gly Phe Val His His Gly Thr Val Le #u Asp Cys Glu Glu Lys                  85  #                 90  #                 95 Asp Trp Asp Phe Ser Met Asn Leu Asn Val Ar #g Asn Val Met Tyr Leu             100       #           105       #           110 Met Ile Lys Ala Phe Leu Pro Lys Met Leu Al #a Gln Lys Ser Gly Asn         115           #       120           #       125 Ile Ile Asn Met Ser Ser Val Ala Ser Ser Va #l Lys Gly Val Val Asn     130               #   135               #   140 Arg Cys Val Tyr Ser Thr Thr Lys Ala Ala Va #l Ile Gly Leu Thr Lys 145                 1 #50                 1 #55                 1 #60 Ser Val Ala Ala Asp Phe Ile Gln Gln Gly Il #e Arg Cys Asn Cys Val                 165   #               170   #               175 Cys Pro Gly Thr Val Asp Thr Pro Ser Leu Gl #n Glu Arg Ile Gln Ala             180       #           185       #           190 Arg Gly Asn Pro Glu Glu Ala Arg Asn Asp Ph #e Leu Lys Arg Gln Lys         195           #       200           #       205 Thr Gly Arg Phe Ala Thr Ala Glu Glu Ile Al #a Met Leu Cys Val Tyr     210               #   215               #   220 Leu Ala Ser Asp Glu Ser Ala Tyr Val Thr Gl #y Asn Pro Val Ile Ile 225                 2 #30                 2 #35                 2 #40 Asp Gly Gly Trp Ser Leu                 245 <210> SEQ ID NO 144 <211> LENGTH: 234 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 144 Gly Thr Ile Gly Leu Tyr Trp Val Gly Ser Il #e Ile Met Ser Val Val   1               5  #                 10  #                 15 Val Phe Val Pro Gly Asn Ile Val Gly Lys Ty #r Gly Thr Arg Ile Cys              20      #             25      #             30 Pro Ala Phe Phe Leu Ser Ile Pro Tyr Thr Cy #s Leu Pro Val Trp Ala          35          #         40          #         45 Gly Phe Arg Ile Tyr Asn Gln Pro Ser Glu As #n Tyr Asn Tyr Pro Ser      50              #     55              #     60 Lys Val Ile Gln Glu Ala Gln Ala Lys Asp Le #u Leu Arg Arg Pro Phe  65                  # 70                  # 75                  # 80 Asp Leu Met Leu Val Val Cys Leu Leu Leu Al #a Thr Gly Phe Cys Leu                  85  #                 90  #                 95 Phe Arg Gly Leu Ile Ala Leu Asp Cys Pro Se #r Glu Leu Cys Arg Leu             100       #           105       #           110 Tyr Thr Gln Phe Gln Glu Pro Tyr Leu Lys As #p Pro Ala Ala Tyr Pro         115           #       120           #       125 Lys Ile Gln Met Leu Ala Tyr Met Phe Tyr Se #r Val Pro Tyr Phe Val     130               #   135               #   140 Thr Ala Leu Tyr Gly Leu Val Val Pro Gly Cy #s Ser Trp Met Pro Asp 145                 1 #50                 1 #55                 1 #60 Ile Thr Leu Ile His Ala Gly Gly Leu Ala Gl #n Ala Gln Phe Ser His                 165   #               170   #               175 Ile Gly Ala Ser Leu His Ala Arg Thr Ala Ty #r Val Tyr Arg Val Pro             180       #           185       #           190 Glu Glu Ala Lys Ile Leu Phe Leu Ala Leu As #n Ile Ala Tyr Gly Val         195           #       200           #       205 Leu Pro Gln Leu Leu Ala Tyr Arg Cys Ile Ty #r Lys Pro Glu Phe Phe     210               #   215               #   220 Ile Lys Thr Lys Ala Glu Glu Lys Val Glu 225                 2 #30 <210> SEQ ID NO 145 <211> LENGTH: 238 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (184) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 145 Met Ser Asn His Asp Pro Arg Gly Cys Thr Ar #g Arg Arg Ala Gln Lys   1               5  #                 10  #                 15 Pro Leu Ala Ile Gln Pro Arg Leu Phe His Al #a Ser Ala Pro Asp Glu              20      #             25      #             30 Gly Thr Gln Gly Thr Leu Lys Gly Thr Gln Ly #s Gly Gly Cys Ile Leu          35          #         40          #         45 Val Gln Cys Gln Ser Glu Gly Gly Ala Ala Gl #y Ala Trp Thr Gly Pro      50              #     55              #     60 Pro Ser Pro Ala Arg Asp Arg Arg Val Arg Pr #o Pro Gly Thr Lys Ala  65                  # 70                  # 75                  # 80 Gln Arg Leu Glu Arg Arg Arg His Val Pro Ar #g Leu His Gly Leu Gly                  85  #                 90  #                 95 Val Gly Gly Cys Glu Val Arg Thr Gly Ile Va #l Ala Arg Ile Ser Gly             100       #           105       #           110 Ser Thr Pro Trp Ala Gly Gly Lys Pro Leu Gl #y Leu His Gly Ala Met         115           #       120           #       125 Gly Glu Ala Gly Ala Gly Asp Thr Gly Cys Cy #s Ala Lys Gly Pro Ser     130               #   135               #   140 Pro Ala Ala Pro Leu Pro Ala Glu Gly Arg Gl #y Gln Gly Ala Gly Pro 145                 1 #50                 1 #55                 1 #60 Gly Gly Leu Val Gly Arg Gly Glu Arg Arg As #p Gln Gln Thr Leu Leu                 165   #               170   #               175 Gly Met Ala Glu Asp Thr Gly Xaa Ser Pro Se #r Arg Pro Ser Ala Pro             180       #           185       #           190 Ala Pro Arg Ala Pro Val Pro Ala Arg Gln Pr #o Leu Pro Arg Ala Arg         195           #       200           #       205 Leu Gly Ala Ala Thr Ala Ile Ser Lys Ser Ar #g Ser Ser Arg Val Ala     210               #   215               #   220 Pro Ala Leu Ala Ala Ala Ile Ser Ala Ser Se #r His Gln Arg 225                 2 #30                 2 #35 <210> SEQ ID NO 146 <211> LENGTH: 207 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (3) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (5) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (9) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (30) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (169) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 146 Ser Thr Xaa Thr Xaa Thr Ile Gly Xaa Ala Gl #y Thr Pro Ala Gly Thr   1               5  #                 10  #                 15 Gly Pro Glu Phe Pro Gly Arg Pro Thr Arg Pr #o Gly Glu Xaa Pro Val              20      #             25      #             30 Asp Phe Ser Lys Gln Tyr Ser Ala Ser Trp Me #t Cys Leu Ser Leu Leu          35          #         40          #         45 Ala Ala Leu Ala Cys Ser Ala Gly Asp Thr Tr #p Ala Ser Glu Val Gly      50              #     55              #     60 Pro Val Leu Ser Lys Ser Ser Pro Arg Leu Il #e Thr Thr Trp Glu Lys  65                  # 70                  # 75                  # 80 Val Pro Val Gly Thr Asn Gly Gly Val Thr Va #l Val Gly Leu Val Ser                  85  #                 90  #                 95 Ser Leu Leu Gly Gly Thr Phe Val Gly Ile Al #a Tyr Phe Leu Thr Gln             100       #           105       #           110 Leu Ile Phe Val Asn Asp Leu Asp Ile Ser Al #a Pro Gln Trp Pro Ile         115           #       120           #       125 Ile Ala Phe Gly Gly Leu Ala Gly Leu Leu Gl #y Ser Ile Val Asp Ser     130               #   135               #   140 Tyr Leu Gly Ala Thr Met Gln Tyr Thr Gly Le #u Asp Glu Ser Thr Gly 145                 1 #50                 1 #55                 1 #60 Met Val Val Asn Ser Pro Thr Asn Xaa Ala Ar #g His Ile Ala Gly Lys                 165   #               170   #               175 Pro Ile Leu Asp Asn Asn Ala Val Asn Leu Ph #e Ser Ser Val Leu Ile             180       #           185       #           190 Ala Leu Leu Leu Pro Thr Ala Ala Trp Gly Ph #e Trp Pro Arg Gly         195           #       200           #       205 <210> SEQ ID NO 147 <211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 147 Met Ser Gln Arg Ala Gly Arg Arg Pro Gly Gl #y Trp Asn Pro Ser Leu   1               5  #                 10  #                 15 Ser Val Val Glu Val Cys Arg Gly Cys Arg Gl #y Thr Gly Pro Leu Pro              20      #             25      #             30 Trp Gly Ala Ser Leu Phe Pro Cys Ser Ala Se #r Pro Leu Phe Pro Leu          35          #         40          #         45 Pro Leu Asn Arg Arg Gly Asp Val His Gly Th #r Leu Gly Gly Arg Met      50              #     55              #     60 Leu Asn Arg Val Glu Cys Arg Asp Gly Val Al #a Ala Ala Trp Leu Cys  65                  # 70                  # 75                  # 80 Leu His Asp Ala Ala Ala Ile Arg Gly Ala Va #l Gly Arg Cys Pro Met                  85  #                 90  #                 95 Trp Thr Gln Pro Thr His Trp Val Leu Leu Le #u Cys Trp Ala Leu His             100       #           105       #           110 Phe Tyr Cys Arg         115 <210> SEQ ID NO 148 <211> LENGTH: 81 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 148 Met Thr Ala His Ser Phe Ala Leu Pro Val Il #e Ile Phe Thr Thr Phe   1               5  #                 10  #                 15 Trp Gly Leu Val Gly Ile Ala Gly Pro Trp Ph #e Val Pro Lys Gly Pro              20      #             25      #             30 Asn Arg Gly Val Ile Ile Thr Met Leu Val Al #a Thr Ala Val Cys Cys          35          #         40          #         45 Tyr Leu Phe Trp Leu Ile Ala Ile Leu Ala Gl #n Leu Asn Pro Leu Phe      50              #     55              #     60 Gly Pro Gln Leu Lys Asn Glu Thr Ile Trp Ty #r Val Arg Phe Leu Trp  65                  # 70                  # 75                  # 80 Glu <210> SEQ ID NO 149 <211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 149 Ala Gln Arg Ala Ala Arg Leu Gly Thr Arg Al #a Pro Ala Ala Pro Ala   1               5  #                 10  #                 15 Ala Arg Pro Cys Ile Leu Pro Gly His Pro Al #a Pro Gly His Asp Gly              20      #             25      #             30 Ala Leu Ile Arg Pro Pro Gly His His Leu Hi #s His Val Leu Gly Pro          35          #         40          #         45 Arg Arg His Arg Gly Pro Trp Phe Val Pro Ly #s Gly Pro Asn Arg Gly      50              #     55              #     60 Val Ile Ile Thr Met Leu Val Ala Thr Ala Va #l Cys Cys Tyr Leu Phe  65                  # 70                  # 75                  # 80 Trp Leu Ile Ala Ile Leu Ala Gln Leu Asn Pr #o Leu Phe Gly Pro Gln                  85  #                 90  #                 95 Leu Lys Asn Glu Thr Ile Trp Tyr Val Arg Ph #e Leu Trp Glu             100       #           105       #           110 <210> SEQ ID NO 150 <211> LENGTH: 135 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 150 Met Thr Leu Glu Glu His Arg Asp Arg Pro Ar #g Leu Gly Met Cys Met   1               5  #                 10  #                 15 Cys Val Cys Ala Cys Val Tyr Ala Cys Met Le #u Met His Val Cys Val              20      #             25      #             30 His Ala Cys Leu Cys Val Cys Val Cys Val Cy #s Val Glu Pro Trp Ser          35          #         40          #         45 Ser Arg Gln Ser Lys Asp Thr Gly Gly Trp Hi #s Met Glu Glu Gln Val      50              #     55              #     60 Thr Pro Pro Ser Leu Ala Gln Leu Lys Ser Gl #y Gln Val Arg Gly Glu  65                  # 70                  # 75                  # 80 Met Gly Glu Gly Arg Gly Glu Lys Gly Glu Gl #u Ala Leu Thr Gly Gly                  85  #                 90  #                 95 Ala Glu Ala Leu Ser Leu Leu Gly Arg Arg Se #r Pro Ser Thr Pro Leu             100       #           105       #           110 Phe Leu Asp Arg Glu Asp Lys Gln Ala Lys As #p Ala Arg Asn Leu Ser         115           #       120           #       125 Ser Thr Val Ala Pro Asp Phe     130               #   135 <210> SEQ ID NO 151 <211> LENGTH: 82 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 151 His Glu Lys Ile Leu Thr Pro Ile Trp Pro Se #r Ser Thr Asp Leu Glu   1               5  #                 10  #                 15 Lys Pro His Glu Met Leu Phe Leu Asn Val Il #e Leu Phe Ser Leu Thr              20      #             25      #             30 Val Phe Thr Leu Ile Ser Thr Ala His Thr Le #u Asp Arg Ala Val Arg          35          #         40          #         45 Ser Asp Trp Leu Leu Leu Val Leu Ile Tyr Al #a Cys Leu Glu Glu Leu      50              #     55              #     60 Ile Pro Glu Leu Ile Phe Asn Leu Tyr Cys Gl #n Gly Asn Ala Thr Leu  65                  # 70                  # 75                  # 80 Phe Phe <210> SEQ ID NO 152 <211> LENGTH: 71 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 152 Pro Ala Asn Lys Ala Gly Ala Ala Ile Glu Al #a Gly Ile Gly Ile Ser   1               5  #                 10  #                 15 Leu Met Val Leu Ser Pro Trp Ala Cys Leu Ph #e Val Val Phe Phe Pro              20      #             25      #             30 Tyr Ile Gln Ser Ser Leu Arg Ser Asp Lys Hi #s Leu Gln Leu Ser Asn          35          #         40          #         45 Ile Leu Pro Thr Pro Ser His His Ile His Le #u Pro Ala Ser Ile Cys      50              #     55              #     60 Ile Gln Leu Arg Ala Gly Asn  65                  # 70 <210> SEQ ID NO 153 <211> LENGTH: 75 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 153 Ala Gly Ser Pro Ala Gly Thr Gly Pro Glu Ph #e Pro Gly Arg Pro Thr   1               5  #                 10  #                 15 Arg Pro Ile Ser Thr His Val Phe Glu Tyr Gl #u Cys Ile Cys Lys Ile              20      #             25      #             30 Pro Arg Phe Met Cys Glu Tyr Val Leu Leu Le #u Tyr Ile Val Leu Leu          35          #         40          #         45 Cys Asn Arg Ser Tyr Ala Val Phe Thr Gln Cy #s Val Leu Arg Ser Ser      50              #     55              #     60 Pro Ile Asp Ser Ser Arg Asn Ala Val Leu Le #u  65                  # 70                  # 75 <210> SEQ ID NO 154 <211> LENGTH: 483 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (194) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (205) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 154 Met Pro Ser Gly Met Ser Ala Ala Val Pro Il #e Ser Gly Leu Leu Asp   1               5  #                 10  #                 15 Leu Ser His Asn Ser Ile Ser Gln Glu Ser Al #a Leu Tyr Leu Leu Glu              20      #             25      #             30 Thr Leu Pro Ser Cys Pro Arg Val Arg Glu Al #a Ser Val Asn Leu Gly          35          #         40          #         45 Ser Glu Gln Ser Phe Arg Ile His Phe Ser Ar #g Glu Asp Gln Ala Gly      50              #     55              #     60 Lys Thr Leu Arg Leu Ser Glu Cys Ser Phe Ar #g Pro Glu His Val Ser  65                  # 70                  # 75                  # 80 Arg Leu Ala Thr Gly Leu Ser Lys Ser Leu Gl #n Leu Thr Glu Leu Thr                  85  #                 90  #                 95 Leu Thr Gln Cys Cys Leu Gly Gln Lys Gln Le #u Ala Ile Leu Leu Ser             100       #           105       #           110 Leu Val Gly Arg Pro Ala Gly Leu Phe Ser Le #u Arg Val Gln Glu Pro         115           #       120           #       125 Trp Ala Asp Arg Ala Arg Val Leu Ser Leu Le #u Glu Val Cys Ala Gln     130               #   135               #   140 Ala Ser Gly Ser Val Thr Glu Ile Ser Ile Se #r Glu Thr Gln Gln Gln 145                 1 #50                 1 #55                 1 #60 Leu Cys Val Gln Leu Glu Phe Pro Arg Gln Gl #u Glu Asn Pro Glu Ala                 165   #               170   #               175 Val Ala Leu Arg Leu Ala His Cys Asp Leu Gl #y Ala His His Ser Leu             180       #           185       #           190 Leu Xaa Gly Gln Leu Met Glu Thr Cys Ala Ar #g Leu Xaa Gln Leu Ser         195           #       200           #       205 Leu Ser Gln Val Asn Leu Cys Glu Asp Asp As #p Ala Ser Ser Leu Leu     210               #   215               #   220 Leu Gln Ser Leu Leu Leu Ser Leu Ser Glu Le #u Lys Thr Phe Arg Leu 225                 2 #30                 2 #35                 2 #40 Thr Ser Ser Cys Val Ser Thr Glu Gly Leu Al #a His Leu Ala Ser Gly                 245   #               250   #               255 Leu Gly His Cys His His Leu Glu Glu Leu As #p Leu Ser Asn Asn Gln             260       #           265       #           270 Phe Asp Glu Glu Gly Thr Lys Ala Leu Met Ar #g Ala Leu Glu Gly Lys         275           #       280           #       285 Trp Met Leu Lys Arg Leu Asp Leu Ser His Le #u Leu Leu Asn Ser Ser     290               #   295               #   300 Thr Leu Ala Leu Leu Thr His Arg Leu Ser Gl #n Met Thr Cys Leu Gln 305                 3 #10                 3 #15                 3 #20 Ser Leu Arg Leu Asn Arg Asn Ser Ile Gly As #p Val Gly Cys Cys His                 325   #               330   #               335 Leu Ser Glu Ala Leu Arg Ala Ala Thr Ser Le #u Glu Glu Leu Asp Leu             340       #           345       #           350 Ser His Asn Gln Ile Gly Asp Ala Gly Val Gl #n His Leu Ala Thr Ile         355           #       360           #       365 Leu Pro Gly Leu Pro Glu Leu Arg Lys Ile As #p Leu Ser Gly Asn Ser     370               #   375               #   380 Ile Ser Ser Ala Gly Gly Val Gln Leu Ala Gl #u Ser Leu Val Leu Cys 385                 3 #90                 3 #95                 4 #00 Arg Arg Leu Glu Glu Leu Met Leu Gly Cys As #n Ala Leu Gly Asp Pro                 405   #               410   #               415 Thr Ala Leu Gly Leu Ala Gln Glu Leu Pro Gl #n His Leu Arg Val Leu             420       #           425       #           430 His Leu Pro Phe Ser His Leu Gly Pro Gly Gl #y Ala Leu Ser Leu Ala         435           #       440           #       445 Arg Pro Trp Met Asp Pro Pro Ile Trp Lys Ar #g Ser Ala Trp Arg Lys     450               #   455               #   460 Thr Thr Trp Leu Glu Gly Ser Cys Val Ser Va #l Trp Ser Ser Arg Cys 465                 4 #70                 4 #75                 4 #80 Ser Asp Arg <210> SEQ ID NO 155 <211> LENGTH: 221 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 155 His Gln Leu Ser Arg Gly Ser Ala Val Gly Ar #g Val Ser Arg Ser Leu   1               5  #                 10  #                 15 Gln Ala Pro Gly Gly Val Asp Ala Trp Leu Gl #n Cys Pro Gly Gly Ser              20      #             25      #             30 His Ser Pro Gly Ala Gly Ser Gly Ala Ala Pr #o Ala Pro Glu Gly Pro          35          #         40          #         45 Thr Pro Thr Ile Gln Pro Ser Gly Pro Arg Tr #p Gly Pro Glu Pro Gly      50              #     55              #     60 Gln Ala Leu Asp Gly Ser Pro His Leu Glu Gl #u Ile Ser Leu Ala Glu  65                  # 70                  # 75                  # 80 Asn Asn Leu Ala Gly Gly Val Leu Arg Phe Cy #s Met Glu Leu Pro Leu                  85  #                 90  #                 95 Leu Arg Gln Ile Asp Leu Val Ser Cys Lys Il #e Asp Asn Gln Thr Ala             100       #           105       #           110 Lys Leu Leu Thr Ser Ser Phe Thr Ser Cys Pr #o Ala Leu Glu Val Ile         115           #       120           #       125 Leu Leu Ser Trp Asn Leu Leu Gly Asp Glu Al #a Ala Ala Glu Leu Ala     130               #   135               #   140 Gln Val Leu Pro Gln Met Gly Arg Leu Lys Ar #g Val Asp Leu Glu Lys 145                 1 #50                 1 #55                 1 #60 Asn Gln Ile Thr Ala Leu Gly Ala Trp Leu Le #u Ala Glu Gly Leu Ala                 165   #               170   #               175 Gln Gly Ser Ser Ile Gln Val Ile Arg Leu Tr #p Asn Asn Pro Ile Pro             180       #           185       #           190 Cys Asp Met Ala Gln His Leu Lys Ser Gln Gl #u Pro Arg Leu Asp Phe         195           #       200           #       205 Ala Phe Phe Asp Asn Gln Pro Gln Ala Pro Tr #p Gly Thr     210               #   215               #   220 <210> SEQ ID NO 156 <211> LENGTH: 89 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 156 Glu Lys Leu Phe Cys Phe Glu Met Leu Leu Il #e Cys Lys Phe Ser Pro   1               5  #                 10  #                 15 Asn Ser Val Pro Pro Glu Thr Cys Ala Ile Le #u Asn Gln Gly Leu Met              20      #             25      #             30 Asp Leu Gly Leu Cys Arg Met Cys Leu Gly As #n Asn Met Phe Ala Gly          35          #         40          #         45 Ser Met Leu Gly Lys Ser His Arg His Ser Pr #o Phe Ser Ile Asn Gln      50              #     55              #     60 Arg His Asn Ala Leu Arg Lys Ala Ala Gly Th #r Pro Ala Gln Lys Ser  65                  # 70                  # 75                  # 80 Leu Gly Ile Val Gln Val Ser Pro Asn                  85 <210> SEQ ID NO 157 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 157 Gly Cys Ala Gly Cys Ala Leu Val Thr Ile Cy #s Leu Gln Ala Val Cys   1               5  #                 10  #                 15 Leu Val Lys Ala Ile Ala Ile Leu His Ser Ar #g Leu Thr Arg Asp Thr              20      #             25      #             30 Met His Cys Gly Arg Pro Gln Gly Pro Leu Pr #o Arg Lys Ala Trp Val          35          #         40          #         45 Leu Ser Arg Phe Pro Pro Thr Glu Thr Ala      50              #     55 <210> SEQ ID NO 158 <211> LENGTH: 48 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 158 Pro Glu Thr Gln Cys Thr Ala Glu Gly Arg Ar #g Asp Pro Cys Pro Glu   1               5  #                 10  #                 15 Lys Pro Gly Tyr Cys Pro Gly Phe Pro Gln Le #u Arg Gln Pro Glu Ile              20      #             25      #             30 Trp Pro Arg Gly Lys Gly Lys Thr Leu His Pr #o Pro Ala Arg His Met          35          #         40          #         45 <210> SEQ ID NO 159 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 159 Ser Glu Ile Gly Glu Asn Arg Pro   1               5 <210> SEQ ID NO 160 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 160 His Asp Thr Asp Ser Phe Ala His   1               5 <210> SEQ ID NO 161 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 161 Ala Leu Arg Lys Ala Ala Gly   1               5 <210> SEQ ID NO 162 <211> LENGTH: 148 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 162 Met Arg Gly Pro Val Cys Gly Phe Ser Leu Va #l Glu Met Leu Leu Ala   1               5  #                 10  #                 15 Leu Ala Leu Gly Leu Met Leu Ile Leu Gly Va #l Thr Gln Ile Ala Leu              20      #             25      #             30 Ser Ser Arg Thr Thr Tyr Ala Ser Gln Ser Al #a Ala Ser Leu Leu Gln          35          #         40          #         45 Asp Asp Ala Arg Phe Ala Leu Gly Lys Leu Il #e Gln Glu Ile Arg Gln      50              #     55              #     60 Ala Gly Met Phe Gly Cys Leu Ser Ala Ala Se #r Ile Ser Asn Ala Pro  65                  # 70                  # 75                  # 80 Ala Gly Phe Asp Arg Pro Ile Gly Trp Ser Th #r Thr Gly Ser Ser Arg                  85  #                 90  #                 95 Ser Leu Thr Leu Val Thr Ala Asp Val Gly Gl #u Gly Gly Ser Lys Pro             100       #           105       #           110 Asp Trp Thr Val Leu Ser Asp Cys Thr Gly Se #r Ala His Ala Tyr Val         115           #       120           #       125 Gly Ser Pro Pro Ala Ala Asn Ala Arg Ala As #n Pro Leu Pro Thr Cys     130               #   135               #   140 Ala Lys Leu Thr 145 <210> SEQ ID NO 163 <211> LENGTH: 137 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 163 Met Gly Tyr Tyr Leu Ser Arg Ser Arg Gln Al #a Gly Met Val Leu Leu   1               5  #                 10  #                 15 Ile Ser Leu Val Phe Leu Leu Leu Leu Ala Le #u Leu Gly Val Ser Ser              20      #             25      #             30 Met Gln Gly Ala Ile Ser Gln Glu Lys Ile Th #r Gly Ser Leu Arg Gln          35          #         40          #         45 Arg Asn Gln Ser Phe Gln Gln Ala Glu Ser Gl #y Leu Arg Leu Gly Glu      50              #     55              #     60 Ser Leu Val Gln Ala Ser Gly Phe Ala Leu Ar #g Pro Cys His Ser Thr  65                  # 70                  # 75                  # 80 Ala Ala Cys Ala Pro Pro Ala Glu Ser Val Se #r Val Val Gly Pro Gly                  85  #                 90  #                 95 Thr Asn Pro Val Ser Thr Val Thr Trp Ile Gl #y Met Lys Asp Gly Val             100       #           105       #           110 Tyr Gly Ile Gln Asn Leu Gly Pro Gly Thr Gl #y Leu Val Asn Ser Arg         115           #       120           #       125 Gln Arg Pro Arg Pro Arg Ser Ile Ala     130               #   135 <210> SEQ ID NO 164 <211> LENGTH: 209 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 164 Glu Asn Glu Ser Thr Lys Glu Pro Ser Leu Le #u Gln Tyr Leu Cys Val   1               5  #                 10  #                 15 Gln Ser Pro Ala Gly Leu Asn Gly Phe Asn Va #l Leu Leu Ser Gly Ser              20      #             25      #             30 Gln Thr Pro Pro Thr Val Gly Pro Ser Ser Gl #y Gln Leu Pro Ser Phe          35          #         40          #         45 Ser Val Pro Cys Met Val Leu Pro Ser Pro Pr #o Leu Gly Pro Phe Pro      50              #     55              #     60 Val Leu Tyr Ser Pro Ala Met Pro Gly Pro Va #l Ser Ser Thr Leu Gly  65                  # 70                  # 75                  # 80 Ala Leu Pro Asn Thr Gly Pro Val Asn Phe Se #r Leu Pro Gly Leu Gly                  85  #                 90  #                 95 Ser Ile Ala Gln Leu Leu Val Gly Pro Thr Al #a Val Val Asn Pro Lys             100       #           105       #           110 Ser Ser Thr Leu Pro Ser Ala Asp Pro Gln Le #u Gln Ser Gln Pro Ser         115           #       120           #       125 Leu Asn Leu Ser Pro Val Met Ser Arg Ser Hi #s Ser Val Val Gln Gln     130               #   135               #   140 Pro Glu Ser Pro Val Tyr Val Gly His Pro Va #l Ser Val Val Lys Leu 145                 1 #50                 1 #55                 1 #60 His Gln Ser Pro Val Pro Val Thr Pro Lys Se #r Ile Gln Arg Thr His                 165   #               170   #               175 Arg Glu Thr Phe Phe Lys Thr Pro Gly Ser Le #u Gly Asp Pro Val Leu             180       #           185       #           190 Lys Arg Arg Glu Arg Asn Asn His Glu Thr Pr #o Ala Arg Pro Arg Gly         195           #       200           #       205 Asp <210> SEQ ID NO 165 <211> LENGTH: 454 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 165 Arg His Glu Arg His Glu Tyr Arg Arg Ala Le #u Asp His Glu Glu Glu   1               5  #                 10  #                 15 Ala Leu Ser Ser Gly Ser Val Gln Glu Ala Gl #u Ala Met Leu Asp Glu              20      #             25      #             30 Pro Gln Glu Gln Ala Glu Gly Ser Leu Thr Va #l Tyr Val Ile Ser Glu          35          #         40          #         45 His Ser Ser Leu Leu Pro Gln Asp Met Met Se #r Tyr Ile Gly Pro Lys      50              #     55              #     60 Arg Thr Ala Val Val Arg Gly Ile Met His Ar #g Glu Ala Phe Asn Ile  65                  # 70                  # 75                  # 80 Ile Gly Arg Arg Ile Val Gln Val Ala Gln Al #a Met Ser Leu Thr Glu                  85  #                 90  #                 95 Asp Val Leu Ala Ala Ala Leu Ala Asp His Le #u Pro Glu Asp Lys Trp             100       #           105       #           110 Ser Ala Glu Lys Arg Arg Pro Leu Lys Ser Se #r Leu Gly Tyr Glu Ile         115           #       120           #       125 Thr Phe Ser Leu Leu Asn Pro Asp Pro Lys Se #r His Asp Val Tyr Trp     130               #   135               #   140 Asp Ile Glu Gly Ala Val Arg Arg Tyr Val Gl #n Pro Phe Leu Asn Ala 145                 1 #50                 1 #55                 1 #60 Leu Gly Ala Ala Gly Asn Phe Ser Val Asp Se #r Gln Ile Leu Tyr Tyr                 165   #               170   #               175 Ala Met Leu Gly Val Asn Pro Arg Phe Asp Se #r Ala Ser Ser Ser Tyr             180       #           185       #           190 Tyr Leu Asp Met His Ser Leu Pro His Val Il #e Asn Pro Val Glu Ser         195           #       200           #       205 Arg Leu Gly Ser Ser Ala Ala Ser Leu Tyr Pr #o Val Leu Asn Phe Leu     210               #   215               #   220 Leu Tyr Val Pro Glu Leu Ala His Ser Pro Le #u Tyr Ile Gln Asp Lys 225                 2 #30                 2 #35                 2 #40 Asp Gly Ala Pro Val Ala Thr Asn Ala Phe Hi #s Ser Pro Arg Trp Gly                 245   #               250   #               255 Gly Ile Met Val Tyr Asn Val Asp Ser Lys Th #r Tyr Asn Ala Ser Val             260       #           265       #           270 Leu Pro Val Arg Val Glu Val Asp Met Val Ar #g Val Met Glu Val Phe         275           #       280           #       285 Leu Ala Gln Leu Arg Leu Leu Phe Gly Ile Al #a Gln Pro Gln Leu Pro     290               #   295               #   300 Pro Lys Cys Leu Leu Ser Gly Pro Thr Ser Gl #u Gly Leu Met Thr Trp 305                 3 #10                 3 #15                 3 #20 Glu Leu Asp Arg Leu Leu Trp Ala Arg Ser Va #l Glu Asn Leu Ala Thr                 325   #               330   #               335 Ala Thr Thr Thr Leu Thr Ser Leu Ala Gln Le #u Leu Gly Lys Ile Ser             340       #           345       #           350 Asn Ile Val Ile Lys Asp Asp Val Ala Ser Gl #u Val Tyr Lys Ala Val         355           #       360           #       365 Ala Ala Val Gln Lys Ser Ala Glu Glu Leu Al #a Ser Gly His Leu Ala     370               #   375               #   380 Ser Ala Phe Val Ala Ser Gln Glu Ala Val Th #r Ser Ser Glu Leu Ala 385                 3 #90                 3 #95                 4 #00 Phe Phe Asp Pro Ser Leu Leu His Leu Leu Ty #r Phe Pro Asp Asp Gln                 405   #               410   #               415 Lys Phe Ala Ile Tyr Ile Pro Leu Phe Leu Pr #o Met Ala Val Pro Ile             420       #           425       #           430 Leu Leu Ser Leu Val Lys Ile Phe Leu Glu Th #r Arg Lys Ser Trp Arg         435           #       440           #       445 Lys Pro Glu Lys Thr Asp     450 <210> SEQ ID NO 166 <211> LENGTH: 66 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 166 Lys Leu Leu Leu Thr Lys Val Glu Gln Lys Le #u Glu Leu Ala Arg Leu   1               5  #                 10  #                 15 Gln Val Asp Thr Ser Gly Ser Lys Glu Phe Gl #y Thr Ser Gly Ile Pro              20      #             25      #             30 Ala Lys Cys Arg Phe Pro Lys Ile Phe Val As #n Thr Asp Asp Thr Tyr          35          #         40          #         45 Glu Glu Leu His Leu Ile Val Tyr Lys Val Th #r Thr Val Phe Leu Pro      50              #     55              #     60 Ala Leu  65 <210> SEQ ID NO 167 <211> LENGTH: 79 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 167 Met Glu Pro Gln Leu Gly Pro Glu Ala Ala Al #a Leu Arg Pro Gly Trp   1               5  #                 10  #                 15 Leu Ala Leu Leu Leu Trp Val Ser Ala Leu Se #r Cys Ser Phe Ser Leu              20      #             25      #             30 Pro Ala Ser Ser Leu Ser Ser Leu Val Pro Gl #n Val Arg Thr Ser Tyr          35          #         40          #         45 Asn Phe Gly Arg Thr Phe Leu Gly Leu Asp Ly #s Cys Asn Ala Cys Ile      50              #     55              #     60 Gly Thr Ser Ile Cys Lys Lys Phe Phe Lys Gl #u Arg Asn Lys Ile  65                  # 70                  # 75 <210> SEQ ID NO 168 <211> LENGTH: 209 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 168 Gln Leu Pro Leu Trp Pro Ser Pro Ala Ser Va #l Gln Pro Arg Val Asp   1               5  #                 10  #                 15 Ser Gln Arg Ala Arg Gly Ser Pro Glu Pro Ly #s Met Glu Pro Gln Leu              20      #             25      #             30 Gly Pro Glu Ala Ala Ala Leu Arg Pro Gly Tr #p Leu Ala Leu Leu Leu          35          #         40          #         45 Trp Val Ser Ala Leu Ser Cys Ser Phe Ser Le #u Pro Ala Ser Ser Leu      50              #     55              #     60 Ser Ser Leu Val Pro Gln Val Arg Thr Ser Ty #r Asn Phe Gly Arg Thr  65                  # 70                  # 75                  # 80 Phe Leu Gly Leu Asp Lys Cys Asn Ala Cys Il #e Gly Thr Ser Ile Cys                  85  #                 90  #                 95 Lys Lys Phe Phe Lys Glu Glu Ile Arg Ser As #p Asn Trp Leu Ala Ser             100       #           105       #           110 His Leu Gly Leu Pro Pro Asp Ser Leu Leu Se #r Tyr Pro Ala Asn Tyr         115           #       120           #       125 Ser Asp Asp Ser Lys Ile Trp Arg Pro Val Gl #u Ile Phe Arg Leu Val     130               #   135               #   140 Ser Lys Tyr Gln Asn Glu Ile Ser Asp Arg Ly #s Ile Cys Ala Ser Ala 145                 1 #50                 1 #55                 1 #60 Ser Ala Pro Lys Thr Cys Ser Ile Glu Arg Va #l Leu Arg Lys Thr Glu                 165   #               170   #               175 Arg Phe Gln Lys Trp Leu Gln Ala Lys Arg Le #u Thr Pro Asp Leu Val             180       #           185       #           190 Gln Asp Cys His Gln Gly Gln Arg Glu Leu Ly #s Phe Leu Cys Met Leu         195           #       200           #       205 Arg <210> SEQ ID NO 169 <211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (96) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (107) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (111) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (115) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (122) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <221> NAME/KEY: SITE <222> LOCATION: (132) <223> OTHER INFORMATION: Xaa equals any of the  #naturally occurring       L-amino acids <400> SEQUENCE: 169 Met Glu Pro Gln Leu Gly Pro Glu Ala Ala Al #a Leu Arg Pro Gly Trp   1               5  #                 10  #                 15 Leu Ala Leu Leu Leu Trp Val Ser Ala Leu Se #r Cys Ser Phe Ser Leu              20      #             25      #             30 Pro Ala Ser Ser Leu Ser Ser Leu Val Pro Gl #n Val Arg Thr Ser Tyr          35          #         40          #         45 Asn Phe Gly Arg Thr Phe Leu Gly Leu Asp Ly #s Cys Asn Ala Cys Ile      50              #     55              #     60 Gly Thr Ser Ile Cys Lys Lys Phe Phe Lys Gl #u Glu Ile Arg Ser Asp  65                  # 70                  # 75                  # 80 Asn Trp Leu Ala Ser His Leu Gly Thr Ala Se #r Arg Phe Pro Leu Xaa                  85  #                 90  #                 95 Ser Tyr Pro Cys Lys Leu Leu Gln Met Ile Xa #a Lys Ile Trp Xaa Pro             100       #           105       #           110 Cys Gly Xaa Leu Leu Thr Gly Gln Gln Xaa Se #r Asn Glu Ile Ser Lys         115           #       120           #       125 Gln Glu Ile Xaa Cys Leu Leu His Pro Pro Pr #o Lys Asn Leu His Ile     130               #   135               #   140 Asp Val 145 <210> SEQ ID NO 170 <211> LENGTH: 97 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 170 Gly Pro Arg Ala Arg Val Gln Gly Phe Ser Gl #y Ala Asp Ile Val Lys   1               5  #                 10  #                 15 Phe Met Ala Leu Gly Ser Met Tyr Leu Val Le #u Thr Leu Ile Val Ala              20      #             25      #             30 Lys Val Leu Arg Gly Ala Glu Pro Cys Cys Gl #y Pro Leu Lys Asn Arg          35          #         40          #         45 Val Leu Arg Pro Cys Pro Leu Pro Val His Cy #s Pro Leu Pro Ile Pro      50              #     55              #     60 Ser Pro Ala Glu Gly Ile Pro Trp Val Ala Ty #r Leu Pro Ile Arg Trp  65                  # 70                  # 75                  # 80 Phe Ile Ser Cys Cys Pro Gly His Cys Ile Gl #n Ile Pro Met Cys Thr                  85  #                 90  #                 95 Ser 

What is claimed is:
 1. An isolated protein comprising amino acid residues 32 to 182 of SEQ ID NO:114.
 2. The isolated protein of claim 1 which comprises amino acid residues 2 to 182 of SEQ ID NO:114.
 3. The isolated protein of claim 1 which comprises amino acid residues 1 to 182 of SEQ ID NO:114.
 4. The protein of claim 1 which further comprises a polypeptide sequence heterologous to SEQ ID NO:
 114. 5. A composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.
 6. An isolated protein produced by the method comprising: (a) expressing the protein of claim 1 by a cell; and (b) recovering said protein.
 7. An isolated protein comprising the amino acid sequence of the secreted portion of the polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No.
 203081. 8. The isolated protein of claim 7 which comprises the amino acid sequence of the complete polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No. 203081, excepting the N-terminal methionine.
 9. The isolated protein of claim 7 which comprises the amino acid sequence of the complete polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No.
 203081. 10. The protein of claim 7 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 11. A composition comprising the protein of claim 7 and a pharmaceutically acceptable carrier.
 12. An isolated protein produced by the method comprising: (a) expressing the protein of claim 7 by a cell; and (b) recovering said protein.
 13. An isolated polypeptide having at least 90% identity to amino acid residues 32 to 182 of SEQ ID NO:114, wherein said polypeptide is expressed in lung lymphomas.
 14. The isolated polypeptide of claim 13, wherein said polypeptide has at least 95% identity to amino acid residues 32 to 182 of SEQ ID NO:
 114. 15. The protein of claim 13 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 16. A composition comprising the protein of claim 13 and a pharmaceutically acceptable carrier.
 17. An isolated protein produced by the method comprising: (a) expressing the protein of claim 13 by a cell; and (b) recovering said protein.
 18. An isolated polypeptide having at least 90% identity to the secreted portion of the polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No. 203081, wherein said polypeptide is expressed in Jung lymphomas.
 19. The isolated polypeptide of claim 18, wherein said polypeptide has at least 95% identity to the polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No.
 203081. 20. The protein of claim 18 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 21. A composition comprising the protein of claim 18 and a pharmaceutically acceptable carrier.
 22. An isolated protein produced by the method comprising: (a) expressing the protein of claim 18 by a cell; and (b) recovering said protein.
 23. An isolated polypeptide having at least 90% identity to amino acid residues 1 to 182 at SEQ ID NO:114, wherein said polypeptide is expressed in lung lymphomas.
 24. The isolated polypeptide of claim 23, wherein said polypeptide has at least 95% identity to amino acid residues 1 to 182 of SEQ ID NO:114.
 25. The protein of claim 23 which comprises a heterologous polypeptide sequence.
 26. A composition comprising the protein of claim 23 and a pharmaceutically acceptable carrier.
 27. An isolated protein produced by the method comprising: (a) expressing the protein of claim 23 by a cell; and (b) recovering said protein.
 28. An isolated polypeptide having at least 90% identity to the complete polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No. 203081, wherein said polypeptide is expressed in lung lymphomas.
 29. The isolated polypeptide of claim 28, wherein said polypeptide has at least 95% identity to the complete polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No.
 203081. 30. The protein of claim 28 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 31. A composition comprising the protein of claim 28 and a pharmaceutically acceptable carrier.
 32. An isolated protein produced by the method comprising: (a) expressing the protein of claim 28 by a cell; and (b) recovering said protein.
 33. An isolated protein consisting of at least 30 contiguous amino acid residues of amino acid residues 32 to 182 of SEQ ID NO:114, wherein said protein is expressed in lung lymphomas.
 34. The isolated protein of claim 33 which consists of at least 50 contiguous amino acid residues of amino acid residues 30 to 182 of SEQ ID NO:114, wherein said protein is expressed in lung lymphomas.
 35. The protein of claim 33 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 36. A composition comprising the protein of claim 33 and a pharmaceutically acceptable carrier.
 37. An isolated protein produced by the method comprising: (a) expressing the protein of claim 33 by a cell; and (b) recovering said protein.
 38. An isolated protein consisting of at least 30 contiguous amino acid residues of The secreted portion of the polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No. 203081, wherein said protein is expressed in lung lymphomas.
 39. The isolated protein of claim 38 which consists of at least 50 contiguous amino acid residues of the secreted portion of the polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No.
 203081. 40. The protein of claim 38 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 41. A composition comprising the protein of claim 38 and pharmaceutically acceptable carrier.
 42. An isolated protein produced by the method comprising: (a) expressing the protein of claim 38 by a cell; and (b) recovering said protein.
 43. An isolated protein consisting of at least 30 contiguous amino acid residues of amino acid residues 1 to 182 of SEQ ID NO:114, wherein said protein is expressed in lung lymphomas.
 44. The isolated protein of claim 43 which consists of at least 50 contiguous amino acid residues of amino acid residues 1 to 182 of SEQ ID NO:114.
 45. The protein of claim 43 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 46. A composition comprising the protein of claim 43 and a pharmaceutically acceptable carrier.
 47. An isolated protein produced by the method comprising: (a) expressing the protein of claim 43 by a cell; and (b) recovering said protein.
 48. An isolated protein consisting of at least 30 contiguous amino acid residues of the complete polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No. 203081, wherein said protein is expressed in lung lymphomas.
 49. The isolated protein of claim 48 which consists of at least 50 contiguous amino acid residues of the complete polypeptide encoded by the HUVDJ43 cDNA contained in ATCC Deposit No.
 203081. 50. The protein of claim 48 which further comprises a polypeptide sequence heterologous to SEQ ID NO:114.
 51. A composition comprising the protein of claim 48 and pharmaceutically acceptable carrier.
 52. An isolated protein produced by the method comprising: (a) expressing the protein of claim 48 by a cell; and (b) recovering said protein. 